Received: 25 August 2023

| Revised: 14 October 2023 | Accepted: 22 October 2023

DOI: 10.1111/cas.16008

ORIGINAL ARTICLE

Antitumor activities against breast cancers by an afucosylated
anti-HER2 monoclonal antibody H2Mab-77-mG2a-f
Tomohiro Tanaka1 | Hiroyuki Suzuki1,2
Yukinari Kato1,2
1
Department of Molecular Pharmacology,
Tohoku University Graduate School of
Medicine, Sendai, Miyagi, Japan2

Department of Antibody Drug
Development, Tohoku University
Graduate School of Medicine, Sendai,
Miyagi, Japan
3

Institute of Microbial Chemistry(BIKAKEN), Numazu, Microbial Chemistry
Research Foundation, Shizuoka, Japan

4

Institute of Microbial Chemistry(BIKAKEN), Laboratory of Oncology,
Microbial Chemistry Research Foundation,
Tokyo, Japan
Correspondence
Hiroyuki Suzuki and Yukinari Kato,
Department of Antibody Drug
Development, Tohoku University
Graduate School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai, Miyagi 980-8575,
Japan.
Email: hiroyuki.suzuki.b4@tohoku.ac.jp
and yukinari.kato.e6@tohoku.ac.jp
Funding information
Japan Agency for Medical Research andDevelopment, Grant/Award Number:
23bm1123027h0001, JP23am0401013,
JP23ama121008 and JP23ck0106730| Tomokazu Ohishi3,4| Mika K. Kaneko1,2 |Abstract
Breast cancer patients with high levels of human epidermal growth factor receptor 2 (HER2) expression have worse clinical outcomes. Anti-HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2-positive breast
cancer. We previously immunized mice with the ectodomain of HER2 to create theanti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa). This was then altered to produce
H2Mab-77-mG2a-f, an afucosylated mouse IgG2a. In the present work, we examined
the reactivity of H2Mab-77-mG2a-f and antitumor effects against breast cancers
in vitro and in vivo. BT-474, an endogenously HER2-expressing breast cancer cellline, was identified by H2Mab-77-mG2a-f with a strong binding affinity (a dissociation constant [KD]: 5.0 × 10−9 M). H2Mab-77-mG2a-f could stain HER2 of breast cancer

tissues in immunohistochemistry and detect HER2 protein in Western blot analysis.
Furthermore, H2Mab-77-mG2a-f demonstrated strong antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for BT-474 cells.
MDA-MB-468, a HER2-negative breast cancer cell line, was unaffected by H2Mab-77mG2a-f. Additionally, in the BT-474-bearing tumor xenograft model, H2Mab-77-mG2a-f
substantially suppressed tumor development when compared with the control mouse
IgG2a mAb. In contrast, the HER2-negative MDA-MB-468-bearing tumor xenograftmodel showed no response to H2Mab-77-mG2a-f. These findings point to the possibility of H2Mab-77-mG2a-f as a treatment regimen by showing that it has antitumor
effects on HER2-positive breast tumors.
KEYWORDS

ADCC, antitumor activity, CDC, HER2, monoclonal antibody

Abbreviations: ADC, antibody–drug conjugate; ADCC, antibody-dependent cellular cytotoxicity; CAR-T, chimeric antigen receptor-T; CDC, complement-dependent cytotoxicity; Fcγ,
Fc-gamma; FDA, US Food and Drug Administration; HER2, human epidermal growth factor receptor 2; IDH1, isocitrate dehydrogenase 1; mAb, monoclonal antibody; NK, natural killer;
PI3K, phosphoinositide 3-kinase; T-DXd, trastuzumab deruxtecan.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.
298

| 	﻿
wileyonlinelibrary.com/journal/cas

Cancer Science. 2024;115:298–309.

1|

I NTRO D U C TI O N

Pertuzumab, a therapeutic antibody authorized in 2012 for
HER2-positive breast cancer, binds to the domain II of HER2, pre-The receptor tyrosine kinase human epidermal growth factor re-venting receptor dimerization with partner receptors and related

ceptor 2 (HER2, also known as ERBB2) is a type I transmembrane

signal transduction.33–35 Pertuzumab, although having ADCC effi-

glycoprotein that controls cell development and survival. HER2 cancacy equal to trastuzumab, does not decrease HER2 shedding. 27,35,36

form homodimers and heterodimers with related family members

A potential mechanism for resistance to anti-HER2 therapy includes

including EGFR, HER3, and HER4. The complexes initiate intracel-alteration of the HER2 ectodomain, which results in a reduction oflular signaling pathways like mitogen-activated protein kinase andanti-HER2 antibody binding affinity.37 Anti-HER2 treatments using

phosphoinositide 3-kinase (PI3K)/Akt. The extracellular domain of

distinct anti-HER2 mAb variants, such as binding epitopes, or in con-

HER2 is composed of four regions, domain I–IV. Unlike other family

junction with chemotherapy might pave the way for the continued

receptors, HER2 extracellular domain basically has an active struc-development of HER2-targeted cancer therapies.

ture; dimer formation with other molecules is possible even without
a ligand.1,2 This structural characteristic is assumed to be the root of
HER2's lack of ligand basis.3 It should be mentioned that HER2 andWe previously created an anti-HER2 mAb (clone H2Mab-77;
mouse IgG1, kappa) by immunizing mice with the ectodomain of

HER2.38 H2Mab-77 could apply to flow cytometry, Western blot-

HER3 interactions create extremely potent mitogenic signals andting, and immunohistochemical analyses. To test its ADCC, com-have been implicated in cancer progression.4–6

plement-dependent cytotoxicity (CDC), and antitumor efficacy inTwenty percent of breast cancer patients have an overexpression
7–10

xenograft models, we also altered H2Mab-77 into a core-fucose-de-Breast tumor inci-

leted and subclass-converted anti-HER2 mAb (H2Mab-77-mG2a-f).

dence and development are correlated with HER2 gene amplification

The activation of natural killer (NK) cells, which is enhanced byand protein overexpression.11 Cell cycle progression, survival, angio-

the antibody's Fc region binding to FcγRIIIa on NK cells, results ingenesis, migration, invasion, and tumorigenesis are all accelerated by

the destruction of target cells, including tumor cells, and facilitatesof HER2, which is linked to a poor prognosis.

12–15

In order to forecast the effectiveness

ADCC.39 A core-fucose deletion in the Fc N-glycan has been shownof anti-HER2 therapy, immunohistochemistry and/or in situ hybridiza-to improve the binding of Fc to FcγRIIIa on effector NK cells.40,41

aberrant activation of HER2.

16

tion are often used to evaluate the HER2 status of breast cancer. The

This technique is also applied to clinically approved afucosylated

development of HER2-targeted medicines has advanced significantly,

mAb targeting CCR4, mogamulizumab (Poteligeo).42 In this work, weand HER2 has grown in popularity as a cancer therapeutic target.17

assessed the antitumor efficacy of H2Mab-77-mG2a-f against breast

Trastuzumab emtansine (T-DM1) and trastuzumab deruxtecancancers.

(T-DXd) are antibody–drug conjugates (ADCs) in which the payload
is attached to trastuzumab, a humanized anti-HER2 monoclonal antibody (mAb).18–20 Trastuzumab has been used to treat HER2-positive
metastatic breast cancer. T-DM1 was authorized for HER2-positive
advanced breast cancer by the US Food and Drug Administration

2|

M ATE R I A L S A N D M E TH O DS

2.1 | Cell lines(FDA) and the European Medicines Agency in 2013, while T-DXd
was approved in the United States in 2019 for HER2-positive met-

Human breast cancer cell lines (BT-474 and MDA-MB-468), a humanastatic or unresectable breast cancer. 21–23 Trastuzumab binds toglioblastoma cell line (LN229), and CHO-K1 were obtained from

HER2 domain IV, causing antibody-dependent cellular cytotoxicity

the American Type Culture Collection. The HER2-overexpressed(ADCC) and antibody-dependent cell phagocytosis. 24–26 The extra-

CHO-K1 (CHO/HER2) and LN229 (LN229/HER2) cell lines were de-cellular domain of HER2 has been seen in breast cancer patients'

scribed previously.38,43,44 CHO-K1 and CHO/HER2 cell lines were

27,28

cultured in Roswell Park Memorial Institute (RPMI)-1640 medium

After connecting to the receptors, the ADCs are internalized by en-(Nacalai Tesque, Inc.). LN229, LN229/HER2, BT-474, MDA-MB-468,

docytosis, where they eventually release their payloads and cause

HEK293T, and HER2-knockout HEK293T (BINDS-23) cell lines werecell damage. As a result, trastuzumab-based ADCs are potent weap-

cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai

ons that combine the dual antitumor action of trastuzumab with aTesque, Inc.). Both media were supplemented with with 10% heat-cytotoxic payload. T-DM1 has been shown to preserve trastuzumab

inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.),

effects such as blocking HER2 ectodomain cleavage, inhibiting

100 μg/mL streptomycin, 100 units/mL penicillin, and 0.25 μg/mL

PI3K/Akt signaling, and engaging immune cells through Fc-gamma

amphotericin B (Nacalai Tesque, Inc.). The cells were maintained in a(Fcγ) receptors, resulting in ADCC. 29 T-DXd is an ADC of the future

humidified incubator at 37°C with 5% CO2 and 95% air atmosphere.

serum, but trastuzumab binding prevents this HER2 cleavage.

that combines deruxtecan and trastuzumab. Due to the bystander
impact of its potent payload on surrounding cells, T-DXd has beenfound to be effective not only on HER2-high tumor cells but also on

2.2 | Animals

HER2-low tumor cells. 22,30 T-DXd is being tested in clinical studies to
see whether it can be used to treat HER2-positive gastric and non-The Institutional Committee for Experiments of the Institute ofsmall cell lung cancer.31,32

Microbial Chemistry (Numazu, Japan) authorized animal studies for

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 299

TANAKA et al.

|

TANAKA et al.

H2Mab-77-mG2a-f's antitumor efficacy (approval numbers 2022-

The binding isotherms were fitted into the built-in, one-site bind-056, 2023-001, and 2023-018). Humane objectives for euthanasia

ing model in GraphPad PRISM 6 (GraphPad Software, Inc.) to calcu-

were established as a loss of original body weight to a point >25%

late the binding affinity.

and/or a maximal tumor size >3000 mm3.

2.3 | Antibodies

2.6 | Western blotting
Western blotting was performed using H2Mab-77-mG2a-f (1 μg/mL)

We cloned the VH cDNA of H2Mab-77 and the CH of mouse IgG2a into
the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation)

and an anti-isocitrate dehydrogenase 1 (IDH1) mAb, RcMab-1 (1 μg/mL), as described previously.38

in order to change the subclass of H2Mab-77 from mouse IgG1 tomouse IgG2a (H2Mab-77-mG2a). Additionally, the pCAG-Neo vector(FUJIFILM Wako Pure Chemical Corporation) was used to clone the

2.7 | Immunohistochemical analysis

VL cDNA of H2Mab-77 and the CL cDNA of the mouse kappa lightchain. The vector for H2Mab-77-mG2a was transfected into BINDS-

After being autoclaved in EnVision FLEX Target Retrieval Solution

09, FUT8-knockout ExpiCHO-S cells. Using Ab-Capcher (ProteNova

High pH (Agilent Technologies, Inc.) for 20 min, paraffin-embed-

Co., Ltd.), H2Mab-77-mG2a-f, an afucosylated variant of the original

ded tissue sections from the breast cancer tissue array (Cat. No.

antibody, was isolated. For the investigation of ADCC, CDC, andT8235721-5, BioChain) were treated with 3% hydrogen peroxide forin vivo antitumor effectiveness, 281-mG2a-f (an afucosylated anti-

15 min at room temperature. After blocking using SuperBlock T20

hamster podoplanin [PDPN] mAb) was employed as an afucosylated(Thermo Fisher Scientific, Inc.), the sections were incubated withreference mouse IgG2a.45–47 Since 281-mG2a-f does not recognize

H2Mab-77-mG2a-f (10 μg/mL) for 60 min and then with the EnVision+

human PDPN, it does not react with human breast cancer cells (BT-Kit for mouse (Agilent Technologies, Inc.) for 30 min. The chromog-

474 and MDA-MB-468).

enic reaction was performed as described previously.38

2.4 | Flow cytometry

2.8 | Antibody-dependent cellular cytotoxicity
reporter bioassay

Cells were harvested using 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). Subsequently, they

The ADCC reporter bioassay was performed using an ADCC

were washed with 0.1% bovine serum albumin (Nacalai Tesque, Inc.)

Reporter Bioassay kit from Promega, following the manufacturer'sin phosphate-buffered saline (PBS), followed by treatment with

instructions. Target cells (12,500 cells per well) were inoculated

H2Mab-77-mG2a-f (1 or 10 μg/mL) for 30 min at 4°C. Then, cells were

into a 96-well white solid plate. H2Mab-77-mG2a-f, H2Mab-77, andincubated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000

281-mG2a-f were serially diluted and added to the target cells. Jurkat

dilution; Cell Signaling Technology, Inc.), and fluorescence wascells stably expressing the human FcγRIIIa receptor, and a nuclear

measured using an SA3800 Cell Analyzer (Sony Corp.).

factor of activated T cells (NFAT) response element driving firefly
luciferase, were used as effector cells. The engineered Jurkat cells

2.5 | Determination of the binding affinity by
enzyme-linked immunosorbent assay (ELISA) andflow cytometry
The serially diluted H2Mab-77 and H2Mab-77-mG2a-f (0.0006–
10 μg/mL) were added to HER2 ectodomain (HER2ec)-immobilized
wells, followed by peroxidase-conjugated anti-mouse immunoglobulins (1:3000 diluted; Agilent Technologies Inc.). Enzymatic reactions
were conducted using 1 Step Ultra TMB (Thermo Fisher Scientific(75,000 cells in 25 μL) were then added and cocultured with antibody-treated target cells at 37°C for 6 h. Luminescence using the
Bio-Glo Luciferase Assay System (Promega) was measured with a
GloMax luminometer (Promega).

2.9 | Antibody-dependent cellular cytotoxicity of
H2Mab-77-mG2a-f
The ADCC activity of H2Mab-77-mG2a-f was measured as describedInc.) followed by the measurement of optical density at 655 nm,

previously.48 Effector cells were obtained from the spleen of female

using an iMark microplate reader (Bio-Rad Laboratories, Inc.).

BALB/c nude mice (Charles River Laboratories). In brief, the spleens

After being suspended in 100 μL of serially diluted H2Mab-77-

were processed through a sterile cell strainer (352360, BD Falcon)mG2a-f (0.0006–10 μg/mL), the cells were incubated with 1:200

to produce single-cell suspensions. A 10-s exposure to ice-cold, dis-of Alexa Fluor 488-conjugated anti-mouse IgG. The SA3800 Celltilled water destroyed erythrocytes. Effector cells were washed with

Analyzer (Sony Corp.) flow cytometer was then used to gather flu-

DMEM and resuspended in DMEM with 10% FBS. Calcein AM (10 μg/

orescence data.

mL) was used to mark the target cells (BT-474 and MDA-MB-468). In

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License300

96-well plates, target cells (2 × 104 cells) were seeded before effector
cells (effector to target ratio, 50:1) and 100 μg/mL of either H2Mab77-mG2a-f or 281-mG2a-f was added. A microplate reader (Power Scan
HT; BioTek Instruments, Inc.) with an excitation wavelength of 485 nm
and an emission wavelength of 538 nm was used to analyze the calcein
release into the medium after a 4.5-h incubation at 37°C.
This is how cytolyticity (% lysis) was determined: % lysis is calculated as (E − S) / (M − S) × 100, where E denotes the fluorescence in
effector and target cell cultures, S denotes the spontaneous fluorescence of only target cells, and M denotes the maximum fluorescence
after treatment with a lysis buffer (10 mM Tris–HCl (pH 7.4), 10 mM
EDTA, and 0.5% Triton X-100).

2.10 | Complement-dependent cytotoxicity of
H2Mab-77-mG2a-f
The following procedure was used to assess how well H2Mab-77mG2a-f induced CDC. With 10 μg/mL Calcein AM, the target cells(BT-474 and MDA-MB-468) were marked. Target cells (2 × 10 4 cells)
were put in 96-well plates with 100 μg/mL of either H2Mab-77mG2a-f or 281-mG2a-f and rabbit complement (final dilution 1:10;
Low-Tox-M Rabbit Complement; Cedarlane Laboratories). The
amount of calcein released into the medium was measured during
4.5-h incubation at 37°C.

2.11 | Antitumor activities of H2Mab-77-mG2a-f in
xenografts of breast cancer
BT-474 and MDA-MB-468 were mixed with DMEM and BD
Biosciences' Matrigel Matrix Growth Factor Reduced. Subcutaneous
injections were then given to the left flanks of BALB/c nude mice. On

F I G U R E 1 Generation of H2Mab-77-mG2a-f (mouse IgG2a) andH2Mab-77-mG2a-f (core-fucose-deleted mouse IgG2a) from theoriginal anti-HER2 mAb, H2Mab-77 (mouse IgG1).

3|

R E S U LT S

3.1 | Development of a core-fucose-deficientanti-HER2 mAb, H2Mab-77-mG2a-f

the seventh post-inoculation day, 100 μg of H2Mab-77-mG2a-f (n = 8)

We previously created an anti-HER2 mAb, H2Mab-77 (mouse IgG1,

or 281-mG2a-f (n = 8) in 100 μL PBS was administered intraperito-

kappa), by immunizing mice with the HER2 ectodomain. Flow cy-

neally. Additional antibody injections were given on days 14 and 21.

tometry, Western blotting, and immunohistochemical tests revealed

The tumor diameter was assessed on days 7, 10, 14, 16, 21, 24, andthat H2Mab-77 has a high binding affinity.38

28 after breast cancer cell implantation. Tumor volumes were cal-In this study, we changed the subclass of antibody to improve

culated in the same manner as previously stated. The weight of theits antitumor efficacy. We altered the subclass of H2Mab-77 frommice was also assessed on days 7, 10, 14, 16, 21, 24, and 28 following

mouse IgG1 to mouse IgG2a by joining the VH chain of H2Mab-77

breast cancer cell inoculation. When the observations were finishedwith the CH chain of mouse IgG2a because mouse IgG1 has no ADCC

on day 28, the mice were sacrificed, and tumor weights were as-

and CDC activity. In addition, utilizing BINDS-09 cells (ExpiCHO-S

sessed following tumor excision.

cells lacking FUT8 fucosyltransferase), an afucosylated mouse IgG2aform of H2Mab-77 (H2Mab-77-mG2a-f) was generated (Figure 1).

2.12 | Statistical analyses
All data are represented as mean ± standard error of the mean (SEM).

3.2 | Analysis of H2Mab-77-mG2a-f reactivity
against breast cancer cells by flow cytometry

Welch's t-test was conducted for ADCC activity, CDC activity, andtumor weight. ANOVA with Sidak's post hoc test was conducted

In a previous study, the original H2Mab-77 (mouse IgG1, kappa)

for tumor volume and mouse weight. GraphPad Prism 8 (GraphPad

was found to be suitable for flow cytometry, Western blotting,

Software, Inc.) was used for all calculations. p < 0.05 was considered

and immunohistochemistry.38 In this study, an afucosylated form

to indicate a statistically significant difference.

of the anti-HER2 mAb, H2Mab-77-mG2a-f, was generated. Similar

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 301

TANAKA et al.

|

TANAKA et al.

F I G U R E 2 Flow cytometric analysis
using H2Mab-77-mG2a-f. (A–D) CHO/
HER2 (A), CHO-K1 (B), HEK293T (C), andHER2-knockout HEK293T (BINDS-23)(D) cells were treated with H2Mab-77mG2a-f or H2Mab-77 (red) or buffer
control (black), followed by Alexa Fluor
488-conjugated anti-mouse IgG. (E, F)
BT-474 (E) and MDA-MB-468 (F) cells
were treated with H2Mab-77-mG2a-f
followed by Alexa Fluor 488-conjugatedanti-mouse IgG. Fluorescence data wereanalyzed using the SA3800 Cell Analyzer.

F I G U R E 3 Determination of thebinding affinity of H2Mab-77-mG2a-f by
ELISA and flow cytometry. (A) HER2ec
was immobilized on immunoplates
and then incubated with the seriallydiluted H2Mab-77 and H2Mab-77mG2a-f (0.0006–10 μg/mL), followedby peroxidase-conjugated anti-mouse
immunoglobulins. Enzymatic reactions
were conducted, and the dissociation
constant (KD) was calculated using
the optical density at 655 nm. (B) The
binding affinity of H2Mab-77-mG2a-f wasdetermined by flow cytometry in LN229/
HER2 and BT-474 cells. Serially diluted
H2Mab-77-mG2a-f was added to the cells,followed by Alexa Fluor 488-conjugatedanti-mouse IgG. Fluorescence data werecollected using the SA3800 Cell Analyzer,
and the KD was calculated.

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License302

to the original H2Mab-77, H2Mab-77-mG2a-f successfully detected
HER2 in HER2-overexpressed Chinese hamster ovary (CHO)-K1(CHO/HER2) cells (Figure 2A), but not in CHO-K1 cells (Figure 2B).
Furthermore, both H2Mab-77 and H2Mab-77-mG2a-f reacted with
HEK293T cells (Figure 2C), but not with HER2-knockout HEK293T(BINDS-23) cells (Figure 2D).
Next, we conducted flow cytometric analysis using H2Mab-77mG2a-f on two breast cancer cell lines, BT-474 and MDA-MB-468.
BT-474 represents the luminal B type of breast cancer with positive
expression of progesterone receptor (PR+), estrogen receptor (ER+),and HER2. In contrast, MDA-MB-468 is a triple-negative breast
cancer lacking expression of PR, ER, and HER2.49 H2Mab-77-mG2a-f
demonstrated recognition of endogenously expressed HER2 in BT474 at 1 or 10 μg/mL, while it showed no response to MDA-MB-468
at either concentration (Figure 2E,F).

3.3 | Determination of the binding
affinity of H2Mab-77-mG2a-f to HER2 by ELISA andflow cytometry
We next evaluated the binding affinity of H2Mab-77 and H2Mab77-mG2a-f to purified HER2ec by ELISA. As shown in Figure 3A,
H2Mab-77 and H2Mab-77-mG2a-f showed comparable disso-

ciation constant (K D) to HER2ec as 3.4 × 10 −10 and 4.9 × 10 −10 M,
respectively. We next investigated the binding affinity of H2Mab77-mG2a-f using flow cytometry. The K D of H2Mab-77-mG2a-f to

LN229/HER2 and BT-474 cells were determined to be 3.4 × 10 −9and 5.0 × 10 −9 M, respectively (Figure 3B). These findings indicatethat H2Mab-77-mG2a-f can effectively recognize HER2 with a highbinding affinity.

3.4 | Western blot and immunohistochemical
analyses using H2Mab-77-mG2a-f

F I G U R E 4 Detection of HER2 by Western blotting using
H2Mab-77-mG2a-f. Cell lysates were electrophoresed andtransferred onto PVDF membranes. After blocking, the PVDF
membranes were incubated with H2Mab-77-mG2a-f (1 μg/mL)(A) or an anti-IDH1 monoclonal antibody (clone RcMab-1, 1 μg/mL) (B), followed by incubation with peroxidase-conjugatedanti-mouse immunoglobulins or peroxidase-conjugated anti-rat
immunoglobulins. (C, D) Detection of HER2 in breast cancer
specimens using immunohistochemical analysis with H2Mab-77mG2a-f. Tissue sections of human breast cancer were incubatedwith H2Mab-77-mG2a-f at a concentration of 10 μg/mL and thentreated with the EnVision+ kit. Scale bar: 100 μm.

Similar to the original H2Mab-77, H2Mab-77-mG2a-f detected theformalin-fixed paraffin-embedded tumor sections for identifying

HER2 band with an estimated 180-kDa band in lysates generated

HER2.

from LN229, LN229/HER2, and BT-474 cells; no band was seen in
MDA-MB-468 cells, which are HER2 negative (Figure 4A). For theinternal control, an anti-IDH1 mAb (clone RcMab-1) was employed(Figure 4B). These findings revealed that H2Mab-77-mG2a-f may be

3.5 | Effector cell activation by H2Mab-77-mG2a-f
against HER2-positive cells

employed in Western blotting to assess HER2 expression in cultured
cells, including breast cancer cells.

The ADCC reporter bioassay is a bioluminescent reporter gene

The H2Mab-77-mG2a-f antibody was then utilized to target

assay to quantify the biological activity of the antibody via

clinical specimens of a human breast cancer tissue array via im-

FcγRIIIa-mediated pathway activation in an ADCC mechanism ofmunohistochemistry analysis. Similar to the original H2Mab-77,

action. To compare the ADCC pathway activation by H2Mab-77-

H2Mab-77-mG2a-f identified HER2 on the plasma membrane of

mG2a-f and H2Mab-77, we treated CHO/HER2 and BT-474 cells

breast cancer tissue (Figure 4C,D). Table S1 summarizes the im-with serially diluted mAbs, and then incubated them with effec-

munohistochemical study of breast cancer. H2Mab-77-mG2a-f

tor Jurkat cells, which express the human FcγRIIIa receptor andeffectively stained HER2 in 14 of 63 cases (22%) of breast can-an NFAT response element driving firefly luciferase reporter.

cer, showing its use in the immunohistochemical investigation of

Furthermore, we also used afucosylated anti-hamster PDPN mAb

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 303

TANAKA et al.

|

TANAKA et al.

F I G U R E 5 Antibody-dependent
cellular cytotoxicity (ADCC) reporter
assay by H2Mab-77-mG2a-f, H2Mab-77,
and 281-mG2a-f in the presence of CHO/
HER2 and BT-474 cells. (A, B) Target cells(CHO/HER2 [A] and BT-474 [B]) weretreated with serially diluted H2Mab-77mG2a-f. (C, D) Target cells (CHO/HER2[C] and BT-474 [D]) were treated with
serially diluted H2Mab-77. (E, F) Target
cells (CHO/HER2 [E] and BT-474 [F]) weretreated with serially diluted 281-mG2a-f(mouse afucosylated IgG2a control).
Then, Jurkat cells stably expressing thehuman FcγRIIIa receptor and an NFAT
response element driving firefly luciferase
reporter were added and cocultured.
Luminescence was measured using the
Bio-Glo Luciferase Assay System.

(mouse IgG2a; 281-mG2a-f) as a control. As shown in Figure 5A,

Then, we looked to see whether H2Mab-77-mG2a-f had CDC

H2Mab-77-mG2a-f dose-dependently activated the effector in theagainst BT-474 and MDA-MB-468 cells. As indicated in Figure 6B,

presence of CHO/HER2 cells (EC 50 ; 35.7 ng/mL; Figure 5A) andH2Mab-77-mG2a-f induced more cytotoxicity (60.5% cytotoxicity)

BT-474 cells (EC 50 ; 44.4 ng/mL; Figure 5B). We confirmed thatin BT-474 cells than control afucosylated mouse IgG2a (26.1% cy-

original H2Mab-77 (Figure 5C,D) and 281-mG2a-f (Figure 5E,F)

totoxicity; p < 0.01). In contrast, the CDC activity of H2Mab-77-

could not activate the effector cells. These results indicated thatmG2a-f was 11.4% cytotoxicity against MDA-MB-468 cells, which

H2Mab-77-mG2a-f exhibits superior ADCC activation activity com-

was comparable to that of 281-mG2a-f (9.3% cytotoxicity). These

pared with H2Mab-77.

encouraging findings show that H2Mab-77-mG2a-f greatly improves both ADCC and CDC activities against HER2-expressing

3.6 | Antibody-dependent cellular cytotoxicity and
CDC by H2Mab-77-mG2a-f against breast cancer
We next tested H2Mab-77-mG2a-f for ADCC against BT-474 cells

breast cancer cells.

3.7 | Antitumor activities of H2Mab-77-mG2a-f
in the mouse xenografts of breast tumor cells(HER2-expressing breast cancer cell line) and MDA-MB-468 cells(HER2-negative breast cancer cell line). As shown in Figure 6A,

Tumor formation was observed on day 7 in 16 mice inoculated

H2Mab-77-mG2a-f demonstrated more ADCC (38.9% cytotoxic-

with either BT-474 or MDA-MB-468 cells. The mice bearingity) against BT-474 cells than 281-mG2a-f, the control afucosylated

breast cancer were divided into two groups: one group received

mouse IgG2a (5.5% cytotoxicity; p < 0.001). In contrast, the ADCC

treatment with H2Mab-77-mG 2a-f, and the other group served as

activity of H2Mab-77-mG2a-f against MDA-MB-468 cells was 9.3%

the control and received 281-mG 2a-f. Intraperitoneal injections

cytotoxicity, which was comparable to that of 281-mG2a-f (10.2%

of H2Mab-77-mG 2a-f (100 μg) and 281-mG 2a-f (100 μg) were ad-cytotoxicity).

ministered to the respective groups on days 7, 14, and 21 after

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License304

in both the BT-474- and MDA-MB-468-bearing xenografts(Figure 7G,H). The appearance of mice treated with H2Mab-77mG 2a-f and 281-mG 2a-f on day 28 after cell inoculation is shownin Figure S1. In summary, these results demonstrate the antitumor effects of H2Mab-77-mG 2a-f administration against HER2positive breast cancer xenografts.

4|

DISCUSSION

Humanized anti-HER2 mAbs like trastuzumab and pertuzumab
have helped patients with HER2-positive breast cancer live
longer.19,21,50,51 Aside from monotherapy with these mAbs, combined treatment with trastuzumab and pertuzumab has been shownto enhance outcomes.52 The membrane-bound p95 generated by
the extracellular cleavage of HER2 by metalloproteinase has kinase
activity in HER2-overexpressing cells. 27 The inhibition of this cleavage by trastuzumab may be one of the factors suppressing proliferative signals. Furthermore, the antitumor effects of trastuzumab isprobably mediated by indirect mechanisms such as ADCC activity.39
In the future, we will look into the impacts of H2Mab-77-mG2a-f on
dimer formation and HER2 shedding to better understand the mechanism of the antitumor action by H2Mab-77-mG2a-f.
The development of significant acquired resistance is a chalF I G U R E 6 Investigation of the antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity(CDC) activities elicited by H2Mab-77-mG2a-f. (A) ADCC elicited by
H2Mab-77-mG2a-f and 281-mG2a-f (control antibodies) targeting
BT-474 and MDA-MB-468 cells. (B) CDC elicited by H2Mab-77mG2a-f and 281-mG2a-f (control antibodies) targeting BT-474 and
MDA-MB-468 cells. Values are presented as the mean ± SEM.
Statistical significance is indicated by asterisks (***p < 0.001,**p < 0.01, n.s., not significant, unpaired t-test).

lenge for the therapeutic use of trastuzumab.53,54 Concerns havebeen raised that even strong ADCs, such as T-DM1, may develop
resistance owing to diminished antigen binding, poor internalization,
lysosomal degradation errors, and accelerated cellular drug clearance by drug-efflux pumps.55 MUC4, a membrane-associated mucin
known to mask membrane proteins, has been shown to inhibit trastuzumab binding to HER2.56 Furthermore, MUC4 acts as an intramembranous ligand and activator of HER2, resulting in the inhibitionof apoptosis in cancer cells.57

cell inoculation. Tumor diameters were measured on days 7, 10,

Antibodies have distinct activities depending on their bind-

14, 16, 21, 24, and 28 following cell inoculation. In the BT-474-

ing epitopes, similar to trastuzumab and pertuzumab. The FDA-

bearing mice, the group treated with H2Mab-77-mG 2a-f exhib-

approved margetuximab and trastuzumab have comparable

ited significantly less tumor growth on days 24 (p < 0.01) and 28

epitopes and binding affinity; however, margetuximab has a(p < 0.001) compared with the group treated with 281-mG 2a-f

greater binding capacity to the ADCC activator FcγRIIIa and a(Figure 7A). The reduction in tumor volume achieved by H2Mab-

lower affinity for the immune activation inhibitor CD32B. 58–60

77-mG 2a-f treatment was 36.6% on day 28. Conversely, there wasPatients with breast cancer who have low-binding FcγRIIIa al-

no difference in tumor growth between the H2Mab-77-mG 2a-f

leles may also benefit from a combination of margetuximab andand 281-mG 2a-f treatment groups in the MDA-MB-468-bearing

anti-HER2 treatments. 61 When used in conjunction with tras-

mice (Figure 7B). Additionally, the tumor weight of the BT-474-

tuzumab, the HER2-targeted humanized mAb 1E11 inhibits thebearing mice treated with H2Mab-77-mG 2a-f was significantly

growth of HER2-expressing gastric tumors by binding to the HER2

lower than that of the mice treated with 281-mG 2a-f (45.1% re-domain IV, which does not overlap with trastuzumab. 62 Therefore,

duction; p < 0.01, Figure 7C). However, no difference in tumor

one of the primary strategies for combating drug resistance is theweight on day 28 was observed between the H2Mab-77-mG 2a-f

development of antibodies with a variety of features, including theand 281-mG 2a-f treatment groups in the MDA-MB-468-bearing
mice (Figure 7D). Figure 7E,F show the resected tumors fromthe H2Mab-77-mG 2a-f- and 281-mG 2a-f-treated groups, respec-

binding epitope. H2Mab-19, 63 H2Mab-41, 64 H2Mab-77, 38 H2Mab-

119, 65 H2Mab-139, 66 H2Mab-181, 67 H2Mab-214, 68 and H2Mab-

250 69 are anti-HER2 mAbs that we have previously established,tively, on day 28 after inoculation of BT-474 and MDA-MB-468

and some of them were proved to have antitumor effects.48,63 In

cells. There were no significant differences in total body weights

our early findings, these mAbs have different epitopes, including

between the H2Mab-77-mG 2a-f- and 281-mG 2a-f-treated groups

HER2 domains I, III, and IV. Trastuzumab and anti-HER2 antibodies

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 305

TANAKA et al.

|

TANAKA et al.

F I G U R E 7 Evaluation of the antitumor
activity of H2Mab-77-mG2a-f in breast
cancer xenograft models. (A, B) BT-474(5 × 106 cells) (A) and MDA-MB-468(5 × 106 cells) (B) were subcutaneouslytransplanted into the left flanks of mice.
On day 7 after transplantation, 100 μgof H2Mab-77-mG2a-f and 281-mG2a-f(control) were intraperitoneally injected
into mice. Additional antibody treatments
were conducted on days 14 and 21. Tumor
diameters were measured on days 7, 10,
14, 16, 21, 24, and 28 after the inoculationof tumor cells. Values are presented as
the mean ± SEM. Statistical significanceis indicated by asterisks (***p < 0.001,**p < 0.01, n.s., not significant, ANOVA,
and Sidak's multiple comparisons test).
(C, D) Tumors of BT-474 (C) and MDAMB-468 (D) xenografts were resectedfrom H2Mab-77-mG2a-f and 281-mG2a-f(control) groups. On day 28, tumor
weight was measured from the excised
xenografts. Values are presented as themean ± SEM. Statistical significance isdenoted by asterisks (**p < 0.01, n.s.,
not significant, unpaired t-test). (E, F)
Resected tumors of BT-474 (E) and MDAMB-468 (F) xenografts from H2Mab-77mG2a-f and 281-mG2a-f (control) groups
on day 28. Scale bar: 1 cm. (G, H) Body
weights of mice inoculated with BT-474(G) and MDA-MB-468 (H) xenograftsin H2Mab-77-mG2a-f and 281-mG2a-f(control) groups were recorded on days
7, 10, 14, 16, 21, 24, and 28. Values aremean ± SEM. No statistical significanceis indicated by n.s. (ANOVA and Sidak's
multiple comparisons test).

that target several epitopes actually have a stronger antitumor impact than standalone therapies.70

Syngeneic mouse models are useful to evaluate the intact
antitumor immune system. A transgenic mouse model using theH2Mab-77-mG2a-f effectively stained HER2 in 14 of 63 cases

mouse mammary tumor virus promoter-controlled HER2 expres-(22%) of breast cancer (Table S1), showing its use in the immuno-sion was developed to evaluate mu4D5 (the murine version ofhistochemical investigation of formalin-fixed paraffin-embedded

trastuzumab).71 The high level of HER2 expression was achieved

tumor sections for identifying HER2. However, the information of

in mammary gland epithelium of young virgin founder mice, which

HER2-positive in the tissue microarray did not necessarily match theresulted in the development of mammary adenocarcinomas in fe-

result of H2Mab-77-mG2a-f staining. Therefore, we speculated thatmale mice with metastasis.71 mu4D5 significantly reduced the inci-

the tissue microarray used in the study may not include the HER2-

dence and growth of mammary adenocarcinomas.71 Furthermore,positive region because HER2-overexpressing tumors are defined by

the tumors could be propagated as allografts in wild-type mice bystrong and complete immunohistochemistry membranous staining

implanting a small piece of tumor into the mammary fat pad. Usingof more than 10% of cells.

the allograft model, they also evaluated T-DM1.72 The modified

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License306

H2Mab-77-mG2a-f demonstrated ADCC and CDC activities de-Microbial Chemistry (approval no. 2022-056, 2023-001, andpending on HER2 expression as well as superior antitumor effects

2023-018).

in xenograft models of HER2-positive breast cancer (Figures 6 and
7). The abovementioned murine model system is useful to com-

ORCID

pare the efficacy of H2Mab-77-mG2a-f with other HER2-targeted

Hiroyuki Suzuki

medicines.

https://orcid.org/0000-0003-2646-5132

Tomokazu Ohishi

Bispecific antibodies targeting HER2 × CD3 (ertumaxomab),

Yukinari Kato

https://orcid.org/0000-0002-9039-4474

https://orcid.org/0000-0001-5385-8201

HER2 × CD16, and HER2 × HER3 (zenocutuzumab: MCLA-128 andMM-111) are being developed in addition to naive antibodies.73–78

REFERENCES

Recently, progress has been made in the creation of bispecific an-

1. Pal SK, Pegram M. Targeting HER2 epitopes. Semin Oncol.
2006;33:386-391.
2. Agus DB, Akita RW, Fox WD, et al. Targeting ligand-activated
ErbB2 signaling inhibits breast and prostate tumor growth. Cancer
Cell. 2002;2:127-137.
3. Garrett TP, McKern NM, Lou M, et al. The crystal structure of a truncated ErbB2 ectodomain reveals an active conformation, poised tointeract with other ErbB receptors. Mol Cell. 2003;11:495-505.
4. Wallasch C, Weiss FU, Niederfellner G, Jallal B, Issing W, Ullrich A.
Heregulin-dependent regulation of HER2/neu oncogenic signalingby heterodimerization with HER3. EMBO J. 1995;14:4267-4275.
5. Alimandi M, Romano A, Curia MC, et al. Cooperative signaling of
ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene. 1995;10:1813-1821.
6. Pinkas-Kramarski R, Soussan L, Waterman H, et al. Diversification ofneu differentiation factor and epidermal growth factor signaling by
combinatorial receptor interactions. EMBO J. 1996;15:2452-2467.
7. Cronin KA, Harlan LC, Dodd KW, Abrams JS, Ballard-Barbash R.
Population-based estimate of the prevalence of HER-2 positive
breast cancer tumors for early stage patients in the US. Cancer
Invest. 2010;28:963-968.
8. Guarneri V, Barbieri E, Dieci MV, Piacentini F, Conte P. Anti-HER2
neoadjuvant and adjuvant therapies in HER2 positive breast cancer. Cancer Treat Rev. 2010;36(Suppl 3):S62-S66.
9. Abd El-Rehim DM, Pinder SE, Paish CE, et al. Expression andco-expression of the members of the epidermal growth factor
receptor (EGFR) family in invasive breast carcinoma. Br J Cancer.
2004;91:1532-1542.
10. Purdie CA, Baker L, Ashfield A, et al. Increased mortality in HER2
positive, oestrogen receptor positive invasive breast cancer: a population-based study. Br J Cancer. 2010;103:475-481.
11. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL.
Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235:177-182.
12. Puglisi F, Fontanella C, Amoroso V, et al. Current challenges in HER2positive breast cancer. Crit Rev Oncol Hematol. 2016;98:211-221.
13. Hayes DF. HER2 and breast cancer–a phenomenal success story. N
Engl J Med. 2019;381:1284-1286.
14. Arteaga CL, Engelman JA. ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics.
Cancer Cell. 2014;25:282-303.
15. Riecke K, Witzel I. Targeting the human epidermal growth factor
receptor family in breast cancer beyond HER2. Breast Care (Basel).
2020;15:579-585.
16. Wolff AC, Hammond ME, Schwartz JN, et al. American Society ofClinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007;25:118-145.
17. Ross JS, Slodkowska EA, Symmans WF, Pusztai L, Ravdin PM,
Hortobagyi GN. The HER-2 receptor and breast cancer: ten years oftargeted anti-HER-2 therapy and personalized medicine. Oncologist.
2009;14:320-368.
18. Cobleigh MA, Vogel CL, Tripathy D, et al. Multinational study of theefficacy and safety of humanized anti-HER2 monoclonal antibody

tibodies that target both the immune checkpoint molecules PD-1/
PD-L1 and HER2, with antitumor effects proven in preclinical animals.79,80 In trastuzumab-resistant cancer models, bispecific antibodies targeting immune checkpoint molecules and HER2 may
be more successful than individual mAb treatments. The use of
H2Mabs to create bispecific antibodies is another option for increasing anti-HER2 treatment. Furthermore, attention has been drawn
to chimeric antigen receptor-T (CAR-T) cell treatment, which possesses both antibody specificity and T cell cytotoxicity.81–83 While
the FDA authorized the first CD19 CAR-T treatment for B-cell lymphoma in 2017, no CAR-T therapeutic targeting HER2 has yet to
be produced.84 In preclinical settings, we previously showed thatanti-PDPN CAR-T cells, in which we incorporated the created anti-PDPN mAb into T cells, had strong antitumor efficacy and may release proinflammatory cytokines.85,86 The application of H2Mab-77
to HER2-targeting CAR-T is expected for future treatment regimen
for HER2-positive breast cancer.
AU T H O R C O N T R I B U T I O N S
Tomohiro Tanaka: Formal analysis; investigation; writing – original draft. Hiroyuki Suzuki: Formal analysis; investigation; writing– original draft. Tomokazu Ohishi: Investigation. Mika K. Kaneko:
Methodology. Yukinari Kato: Funding acquisition; project administration; writing – review and editing.
AC K N OW L E D G M E N T S
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.
F U N D I N G I N FO R M AT I O NThis research was supported in part by the Japan Agency for
Medical Research and Development (AMED) under grant numbers

JP23ama121008(toY.K.),

JP23am0401013(toY.K.),

23bm1123027h0001 (to Y.K.), and JP23ck0106730 (to Y.K.).
E T H I C S S TAT E M E N T
Approval of the research protocol: N/A.
Informed Consent: N/A.
Registry and the Registration No. of the study/trial: N/A.
Animal Studies: Animal experiments were approved by theInstitutional Committee for Experiments of the Institute of

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 307

TANAKA et al.

19.

20.

21.

22.

23.

24.
25.

26.

27.

28.

29.

30.

31.
32.

33.

34.

35.

36.

37.
38.

|
in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease.
J Clin Oncol. 1999;17:2639-2648.
Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy
plus a monoclonal antibody against HER2 for metastatic breast
cancer that overexpresses HER2. N Engl J Med. 2001;344:783-792.
Vogel CL, Cobleigh MA, Tripathy D, et al. Efficacy and safetyof trastuzumab as a single agent in first-line treatment of
HER2-overexpressing metastatic breast cancer. J Clin Oncol.
2002;20:719-726.
Amiri-Kordestani L, Blumenthal GM, Xu QC, et al. FDA approval: ado-trastuzumab emtansine for the treatment of patientswith HER2-positive metastatic breast cancer. Clin Cancer Res.
2014;20:4436-4441.
Nakada T, Sugihara K, Jikoh T, Abe Y, Agatsuma T. The latest research and development into the antibody-drug conjugate, [fam-]
trastuzumab deruxtecan (DS-8201a), for HER2 cancer therapy.
Chem Pharm Bull (Tokyo). 2019;67:173-185.
Zheng Y, Zou J, Sun C, Peng F, Peng C. Ado-tratuzumab emtansine
beyond breast cancer: therapeutic role of targeting other HER2positive cancers. Front Mol Biosci. 2023;10:1165781.
Hudis CA. Trastuzumab–mechanism of action and use in clinicalpractice. N Engl J Med. 2007;357:39-51.
Cho HS, Mason K, Ramyar KX, et al. Structure of the extracellular region of HER2 alone and in complex with the Herceptin fab.
Nature. 2003;421:756-760.
Shi Y, Fan X, Deng H, et al. Trastuzumab triggers phagocytic killingof high HER2 cancer cells in vitro and in vivo by interaction with Fcγ
receptors on macrophages. J Immunol. 2015;194:4379-4386.
Molina MA, Codony-Servat J, Albanell J, Rojo F, Arribas J, Baselga
J. Trastuzumab (herceptin), a humanized anti-Her2 receptor monoclonal antibody, inhibits basal and activated Her2 ectodomain
cleavage in breast cancer cells. Cancer Res. 2001;61:4744-4749.
Loo L, Capobianco JA, Wu W, et al. Highly sensitive detection of
HER2 extracellular domain in the serum of breast cancer patientsby piezoelectric microcantilevers. Anal Chem. 2011;83:3392-3397.
Junttila TT, Li G, Parsons K, Phillips GL, Sliwkowski MX.
Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action of
trastuzumab and efficiently inhibits growth of lapatinib insensitive
breast cancer. Breast Cancer Res Treat. 2011;128:347-356.
Ogitani Y, Hagihara K, Oitate M, Naito H, Agatsuma T. Bystander
killing effect of DS-8201a, a novel anti-human epidermal growth
factor receptor 2 antibody-drug conjugate, in tumors with humanepidermal growth factor receptor 2 heterogeneity. Cancer Sci.
2016;107:1039-1046.
Li BT, Smit EF, Goto Y, et al. Trastuzumab deruxtecan in HER2mutant non-small-cell lung cancer. N Engl J Med. 2022;386:241-251.
Shitara K, Bang YJ, Iwasa S, et al. Trastuzumab deruxtecan inpreviously treated HER2-positive gastric cancer. N Engl J Med.
2020;382:2419-2430.
Harbeck N, Beckmann MW, Rody A, et al. HER2 dimerization inhibitor pertuzumab–mode of action and clinical data in breast cancer.
Breast Care. 2013;8:49-55.
Barthélémy P, Leblanc J, Goldbarg V, Wendling F, Kurtz JE.
Pertuzumab: development beyond breast cancer. Anticancer Res.
2014;34:1483-1491.
Franklin MC, Carey KD, Vajdos FF, Leahy DJ, de Vos AM, Sliwkowski
MX. Insights into ErbB signaling from the structure of the ErbB2pertuzumab complex. Cancer Cell. 2004;5:317-328.
Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med.
2000;6:443-446.
Hou Y, Nitta H, Li Z. HER2 Intratumoral heterogeneity in breast
cancer, an evolving concept. Cancers (Basel). 2023;15:15.
Itai S, Fujii Y, Kaneko MK, et al. H2Mab-77 is a sensitive and specificanti-HER2 monoclonal antibody against breast cancer. Monoclon
Antib Immunodiagn Immunother. 2017;36:143-148.

TANAKA et al.

39. Tsao LC, Force J, Hartman ZC. Mechanisms of therapeutic antitumor monoclonal antibodies. Cancer Res. 2021;81:4641-4651.
40. Shinkawa T, Nakamura K, Yamane N, et al. The absence of fucose
but not the presence of galactose or bisecting N-acetylglucosamineof human IgG1 complex-type oligosaccharides shows the criticalrole of enhancing antibody-dependent cellular cytotoxicity. J Biol
Chem. 2003;278:3466-3473.
41. Kaneko MK, Ohishi T, Nakamura T, et al. Development of core-fucose-deficient humanized and chimeric anti-human podoplanin antibodies. Monoclon Antib Immunodiagn Immunother.
2020;39:167-174.
42. Subramaniam JM, Whiteside G, McKeage K, Croxtall JC.
Mogamulizumab: first global approval. Drugs. 2012;72:1293-1298.
43. Fujii Y, Kaneko MK, Kato Y. MAP tag: a novel tagging system forprotein purification and detection. Monoclon Antib Immunodiagn
Immunother. 2016;35:293-299.
44. Fujii Y, Kaneko MK, Ogasawara S, et al. Development of RAP tag,a novel tagging system for protein detection and purification.
Monoclon Antib Immunodiagn Immunother. 2017;36:68-71.
45. Ikota H, Nobusawa S, Arai H, et al. Evaluation of IDH1 status in
diffusely infiltrating gliomas by immunohistochemistry using anti-mutant and wild type IDH1 antibodies. Brain Tumor Pathol.
2015;32:237-244.
46. Kato Y. Specific monoclonal antibodies against IDH1/2 mutations
as diagnostic tools for gliomas. Brain Tumor Pathol. 2015;32:3-11.
47. Nanamiya R, Suzuki H, Takei J, et al. Development of monoclonal antibody 281-mG(2a)-f against golden hamster podoplanin.
Monoclon Antib Immunodiagn Immunother. 2022;41:311-319.
48. Kato Y, Ohishi T, Takei J, et al. An anti-human epidermal growth
factor receptor 2 monoclonal antibody H2Mab-19 exerts antitumor activity in mouse colon cancer xenografts. Monoclon AntibImmunodiagn Immunother. 2020;39:123-128.
49. Song KH, Trudeau T, Kar A, Borden MA, Gutierrez-Hartmann A.
Ultrasound-mediated delivery of siESE complexed with microbubbles attenuates HER2+/− cell line proliferation and tumor
growth in rodent models of breast cancer. Nanotheranostics.
2019;3:212-222.
50. Valabrega G, Montemurro F, Aglietta M. Trastuzumab: mechanism of action, resistance and future perspectives in HER2overexpressing breast cancer. Ann Oncol. 2007;18:977-984.
51. Amiri-Kordestani L, Wedam S, Zhang L, et al. First FDA approval ofneoadjuvant therapy for breast cancer: pertuzumab for the treatment of patients with HER2-positive breast cancer. Clin Cancer Res.
2014;20:5359-5364.
52. Swain SM, Kim SB, Cortés J, et al. Pertuzumab, trastuzumab,
and docetaxel for HER2-positive metastatic breast cancer(CLEOPATRA study): overall survival results from a randomised,double-blind, placebo-controlled, phase 3 study. Lancet Oncol.
2013;14:461-471.
53. Derakhshani A, Rezaei Z, Safarpour H, et al. Overcoming trastuzumab resistance in HER2-positive breast cancer using combination therapy. J Cell Physiol. 2020;235:3142-3156.
54. Vivekanandhan S, Knutson KL. Resistance to trastuzumab. Cancers(Basel). 2022;14:14.
55. Khoury R, Saleh K, Khalife N, et al. Mechanisms of resistance toantibody-drug conjugates. Int J Mol Sci. 2023;24:9674.
56. Nagy P, Friedländer E, Tanner M, et al. Decreased accessibility andlack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4expressing breast cancer cell line. Cancer Res. 2005;65:473-482.
57. Carraway KL, Perez A, Idris N, et al. Muc4/sialomucin complex, theintramembrane ErbB2 ligand, in cancer and epithelia: to protect andto survive. Prog Nucleic Acid Res Mol Biol. 2002;71:149-185.
58. Royce M, Osgood CL, Amatya AK, et al. FDA approval summary:Margetuximab plus chemotherapy for advanced or metastatic
HER2-positive breast cancer. Clin Cancer Res. 2022;28:1487-1492.
59. Nordstrom JL, Gorlatov S, Zhang W, et al. Anti-tumor activity andtoxicokinetics analysis of MGAH22, an anti-HER2 monoclonal

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

308

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

antibody with enhanced Fcγ receptor binding properties. Breast
Cancer Res. 2011;13:R123.
Rugo HS, Im SA, Cardoso F, et al. Efficacy of Margetuximab vstrastuzumab in patients with pretreated ERBB2-positive advanced
breast cancer: a phase 3 randomized clinical trial. JAMA Oncol.
2021;7:573-584.
Mandó P, Rivero SG, Rizzo MM, Pinkasz M, Levy EM. Targeting
ADCC: a different approach to HER2 breast cancer in the immunotherapy era. Breast. 2021;60:15-25.
Ko BK, Lee SY, Lee YH, et al. Combination of novel HER2targeting antibody 1E11 with trastuzumab shows synergistic
antitumor activity in HER2-positive gastric cancer. Mol Oncol.
2015;9:398-408.
Takei J, Kaneko MK, Ohishi T, Kawada M, Harada H, Kato Y. H2Mab19, an anti-human epidermal growth factor receptor 2 monoclonal
antibody exerts antitumor activity in mouse oral cancer xenografts.
Exp Ther Med. 2020;20:846-853.
Tateyama N, Asano T, Ohishi T, et al. An anti-HER2 monoclonal
antibody H2Mab-41 exerts antitumor activities in mouse xenograft model using dog HER2-overexpressed cells. Monoclon AntibImmunodiagn Immunother. 2021;40:184-190.
Yamada S, Itai S, Nakamura T, et al. Establishment of H(2)Mab-119,
an anti-human epidermal growth factor receptor 2 monoclonal
antibody, against pancreatic cancer. Monoclon Antib Immunodiagn
Immunother. 2017;36:287-290.
Kaneko MK, Yamada S, Itai S, Kato Y. Development of an anti-HER2
monoclonal antibody H2Mab-139 against colon cancer. Monoclon
Antib Immunodiagn Immunother. 2018;37:59-62.
Takei J, Asano T, Tanaka T, et al. Development of a novel anti-HER2
monoclonal antibody H(2)Mab-181 for gastric cancer. Monoclon
Antib Immunodiagn Immunother. 2021;40:168-176.
Arimori T, Mihara E, Suzuki H, et al. Locally misfolded HER2 expressed on cancer cells is a promising target for development ofcancer-specific antibodies. Cell Press Community Review. 2023.
doi:10.2139/ssrn.4565236. dx.​doi.​org
Suzuki H, Kaneko MK, Kato Y. A cancer-specific monoclonal antibody against HER2 for breast cancers Preprint. 2023. doi:10.20944/
preprints202309.0906.v1
Spiridon CI, Ghetie MA, Uhr J, et al. Targeting multiple her-2 epitopes with monoclonal antibodies results in improved antigrowth
activity of a human breast cancer cell line in vitro and in vivo. Clin
Cancer Res. 2002;8:1720-1730.
Finkle D, Quan ZR, Asghari V, et al. HER2-targeted therapy reduces
incidence and progression of midlife mammary tumors in female
murine mammary tumor virus huHER2-transgenic mice. Clin Cancer
Res. 2004;10:2499-2511.
Lewis Phillips GD, Li G, Dugger DL, et al. Targeting HER2-positive
breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug
conjugate. Cancer Res. 2008;68:9280-9290.
Turini M, Chames P, Bruhns P, Baty D, Kerfelec B. A FcγRIII-engaging
bispecific antibody expands the range of HER2-expressing breast
tumors eligible to antibody therapy. Oncotarget. 2014;5:5304-5319.
Diermeier-Daucher S, Ortmann O, Buchholz S, Brockhoff G.
Trifunctional antibody ertumaxomab: non-immunological effects on Her2 receptor activity and downstream signaling. MAbs.
2012;4:614-622.

75. Haense N, Atmaca A, Pauligk C, et al. A phase I trial of the trifunctional anti Her2 × anti CD3 antibody ertumaxomab in patients withadvanced solid tumors. BMC Cancer. 2016;16:420.
76. Schram AM, Odintsov I, Espinosa-Cotton M, et al. Zenocutuzumab, aHER2xHER3 bispecific antibody, is effective therapy for tumors driven
by NRG1 gene rearrangements. Cancer Discov. 2022;12:1233-1247.
77. Fontana E, Torga G, Fostea R, et al. Sustained tumor regression with
Zenocutuzumab, a bispecific antibody targeting human epidermal
growth factor receptor 2/human epidermal growth factor receptor 3 signaling, in NRG1 fusion-positive, estrogen receptor-positive
breast cancer after progression on a cyclin-dependent kinase 4/6
inhibitor. JCO Precis Oncol. 2022;6:e2100446.
78. McDonagh CF, Huhalov A, Harms BD, et al. Antitumor activity of anovel bispecific antibody that targets the ErbB2/ErbB3 oncogenic
unit and inhibits heregulin-induced activation of ErbB3. Mol Cancer
Ther. 2012;11:582-593.
79. Chen YL, Cui Y, Liu X, et al. A bispecific antibody targeting HER2
and PD-L1 inhibits tumor growth with superior efficacy. J Biol
Chem. 2021;297:101420.
80. Gu CL, Zhu HX, Deng L, et al. Bispecific antibody simultaneously
targeting PD1 and HER2 inhibits tumor growth via direct tumor cellkilling in combination with PD1/PDL1 blockade and HER2 inhibition. Acta Pharmacol Sin. 2022;43:672-680.
81. Boulch M, Cazaux M, Loe-Mie Y, et al. A cross-talk between CAR
T cell subsets and the tumor microenvironment is essential for sustained cytotoxic activity. Sci Immunol. 2021;6:6.
82. Sureda A, Lugtenburg PJ, Kersten MJ, et al. Cellular therapy in lymphoma. Hematol Oncol. 2023.
83. Shin MH, Oh E, Kim Y, et al. Recent advances in CAR-based solid
tumor immunotherapy. Cell. 2023;12:12.
84. Mullard A. FDA approves first CAR T therapy. Nat Rev Drug Discov.
2017;16:669.
85. Shiina S, Ohno M, Ohka F, et al. CAR T cells targeting Podoplanin
reduce orthotopic glioblastomas in mouse brains. Cancer Immunol
Res. 2016;4:259-268.
86. Ishikawa A, Waseda M, Ishii T, Kaneko MK, Kato Y, Kaneko S.
Improved anti-solid tumor response by humanized anti-podoplaninchimeric antigen receptor transduced human cytotoxic T cells in an
animal model. Genes Cells. 2022;27:549-558.

S U P P O R T I N G I N FO R M AT I O N
Additional supporting information can be found online in the
Supporting Information section at the end of this article.

How to cite this article: Tanaka T, Suzuki H, Ohishi T,
Kaneko MK, Kato Y. Antitumor activities against breast
cancers by an afucosylated anti-HER2 monoclonal antibody
H2Mab-77-mG2a-f. Cancer Sci. 2024;115:298-309. doi:10.1111/
cas.16008

13497006, 2024, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16008 by Cochrane Japan, Wiley Online Library on [29/01/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 309

TANAKA et al.