EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 846-853, 2020

846

H2Mab‑19, an anti‑human epidermal growth factor
receptor 2 monoclonal antibody exerts antitumor
activity in mouse oral cancer xenografts
JUNKO TAKEI1,2, MIKA KATO KANEKO1, TOMOKAZU OHISHI3, MANABU KAWADA3,HIROYUKI HARADA2 and YUKINARI KATO1,4
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Miyagi 980‑8575; 2Department of Oral and Maxillofacial Surgery, Graduate School of Medical and
Dental Sciences, Tokyo Medical and Dental University, Tokyo 113‑8510; 3Institute of Microbial Chemistry (BIKAKEN),
Numazu, Microbial Chemistry Research Foundation, Numazu‑shi, Shizuoka 410‑0301;
4
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi 980‑8575, Japan
Received November 8, 2019; Accepted February 28, 2020
DOI: 10.3892/etm.2020.8765
Abstract. Human epidermal growth factor receptor 2 (HER2)
is reported to be overexpressed in breast cancers and is associated with poor clinical outcome. Trastuzumab is a humanizedanti‑HER2 antibody that offers significant survival benefits topatients with HER2‑overexpressing breast cancer. In this study,
a novel anti‑HER2 monoclonal antibody (mAb), H2Mab‑19(IgG2b, kappa) was developed. Antibody‑dependent cellularcytotoxicity (ADCC), complement‑dependent cytotoxicity(CDC), and antitumor activity of H2Mab‑19 were investigatedusing both breast cancer and oral cancer cell lines. H2Mab‑19
demonstrated cytotoxicity in BT‑474 (a human breast cancer
cell line) and HSC‑2 or SAS (human oral cancer cell lines).
H2Mab‑19 also possessed both ADCC and CDC activity against
BT‑474, HSC‑2, and SAS cell lines. In comparison to control
mouse IgG, H2Mab‑19 significantly reduced tumor development in BT‑474, HSC‑2, and SAS xenografts. Collectively,
these results suggest that treatment with H2Mab‑19 may be a

Correspondence to: Professor Yukinari Kato, New Industry

Creation Hatchery Center, Tohoku University, 2‑1 Seiryo‑machi,
Aoba‑ku, Sendai, Miyagi 980‑8575, JapanE‑mail: yukinarikato@med.tohoku.ac.jp

Abbreviations:

EGFR, epidermal growth factor receptor;
HER, human epidermal growth factor receptor; OSCC, oral
squamous cell carcinoma; mAbs, monoclonal antibodies;
ADCC,
antibody‑dependent
cellularcytotoxicity;
CDC,
complement‑dependent cytotoxicity; HNC, head and neck cancer;
PD‑1, programmed cell death‑1; VEGF, vascular endothelial
growth factor; DMEM, Dulbecco's modified Eagle's medium;
ELISA, enzyme‑linked immunosorbent assay; FBS, fetal bovine
serum; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum
albumin; PBS, phosphate‑buffered saline

Key words: HSC‑2, monoclonal antibody, antitumor activity

useful therapy for patients with HER2‑expressing breast andoral cancers.
Introduction
The overexpression of human epidermal growth factor
receptor 2 (HER2) is reported in breast (1,2), gastric (3),pancreatic (4), lung (5), and colorectal cancers (6). This
expression is associated with poor clinical outcomes inpatients with HER2‑positive breast cancer (1,2). Humanizedanti‑HER2 monoclonal antibodies (mAbs) trastuzumab andpertuzumab have been used in the treatment of HER2‑positive
breast cancer (7‑9). Treatment with trastuzumab resulted insignificant survival benefits these patients (10). In comparison
to trastuzumab monotherapy, the combination of trastuzumab
and pertuzumab with chemotherapy has led to significant
improvements in overall survival (11).
Trastuzumab deruxtecan (DS‑8201), a recently developed
drug, is comprised of three components, a novel enzyme‑cleavable linker, and a topoisomerase I inhibitor (12). Even in
low‑HER2‑expressing tumors, DS‑8201 shows antitumor
activity. This drug has several innovative features: i) a highly
potent, novel payload with a high drug‑to‑antibody ratio,ii) good homogeneity, iii) a tumor‑selective cleavable linker,iv) a stable linker‑payload in circulation, and v) a cytotoxic
agent with a short in vivo half‑life in vivo (13). Furthermore,
the cytotoxic payload can exert a bystander effect (13).
The novel anti‑HER2 mAb (H2Mab‑19) developed in thisstudy was investigated for its antitumor activities in mouse
xenograft models of breast and oral cancers. These properties
have not been previously investigated with regard to HER2
expression.
Materials and methods
Cell lines. Oral squamous carcinoma cell lines including
Ca9‑22 (derived from gingiva), HO‑1‑u‑1 (mouth floor),
HSC‑2 (oral cavity), and SAS (tongue) were obtained from

TAKEI et al: AN ANTI-HER2 mAb FOR ORAL CANCERS

the Japanese Collection of Research Bioresources Cell Bank
(Osaka, Japan). LN229 (glioblastoma cell line), MDA‑MB‑468(breast cancer), BT‑474 (breast cancer), and P3U1 (mouse
myeloma) were obtained from the American Type Culture
Collection. LN229/HER2 cells were established in a previous
study (14). P3U1 cells were cultured in RPMI‑1640 medium(Nacalai Tesque, Inc., Kyoto, Japan). LN229, LN229/HER2,
MDA‑MB‑468, BT‑474, Ca9‑22, HO‑1‑u‑1, HSC‑2, andSAS were cultured in Dulbecco's modified Eagle's medium(DMEM; Nacalai Tesque, Inc.) supplemented with 10%
heat‑inactivated fetal bovine serum (Thermo Fisher ScientificInc.), 100 units/ml of penicillin, 100 µg/ml streptomycin, and25 µg/ml amphotericin B (Nacalai Tesque, Inc.) at 37̊C in ahumidified atmosphere containing 5% CO2.
Animals. All animal experiments were performed in accordance with relevant guidelines and regulations to minimize
animal suffering and distress in the laboratory. Animal experiments for hybridoma production were approved by the Animal
Care and Use Committee of Tohoku University (permit
no. 2016MdA‑153). Animal health was monitored daily.
Animal studies for Antibody‑Dependent Cellular Cytotoxicity
were approved by the institutional committee for experimentsof the Institute of Microbial Chemistry (permit no. 2019‑066).
Animal studies for antitumor activity were approved by theinstitutional committee for experiments of the Institute ofMicrobial Chemistry (permit no. 2019‑014). Mice were monitored for health and weight every 3 or 4 days. Experiment
duration was three weeks. A bodyweight loss exceeding 25%
and a maximum tumor size exceeding 3,000 mm3 were identified as humane endpoints. Mice were euthanized by cervicaldislocation, and the death was verified by respiratory arrest
and cardiac arrest.
Hybridoma production. One four‑week‑old female BALB/cmouse was purchased from CLEA Japan and housed under
specific pathogen‑free conditions. Anti‑HER2 hybridoma
cells were produced as described previously (14). Briefly,
the BALB/c animal was immunized by intraperitoneal (i.p.)
administration of 100 µg recombinant HER2 extracellulardomain along with Imject Alum (Thermo Fisher ScientificInc.). After several additional immunizations, a booster dose
was administered i.p. 2 days before harvesting spleen cells.
Mice were euthanized by cervical dislocation, and the death
was verified by respiratory arrest and cardiac arrest. Spleencells were then fused with P3U1 cells using PEG1500 (Roche
Diagnostics, Indianapolis, IN, USA). The resulting hybridoma
cells were grown in RPMI medium supplemented withhypoxanthine, aminopterin, and thymidine selection medium(Thermo Fisher Scientific, Inc.). Culture supernatants werescreened using enzyme‑linked immunosorbent assays withrecombinant HER2 extracellular domain. mAbs were purified from the supernatants of hybridoma cells and cultured
in Hybridoma‑SFM medium (Thermo Fisher Scientific, Inc.)
using Protein G Sepharose 4 Fast Flow (GE Healthcare UKLtd.).
Flow cytometry. Hybridoma cells were harvested by brief
exposure to 0.25% trypsin/1‑mM ethylenediaminetetraacetic
acid (EDTA; Nacalai Tesque, Inc.). After washing with 0.1%

847

bovine serum albumin in phosphate‑buffered saline (PBS),
cells were treated with 1 µg/ml anti‑HER2 (H2Mab‑19) for
30 min at 4̊C and subsequently with Alexa Fluor 488‑conjugated anti‑mouse IgG (1:1,000; Cell Signaling Technology,Inc.). Fluorescence microscopy data were collected using an
EC800 Cell Analyzer (Sony Corp.).
Immunohistochemical analyses for formalin‑fixed paraffin‑​embedded (FFPE) tissues. Histologic sections (catalog
no. T8235721‑5; lot no. B104066; BioChain Institute Inc.) werepurchased in this study. Four‑µm histologic sections fromparaffin blocks of resected xenografts were also produced.
These sections were deparaffinized in xylene, then rehydrated
and autoclaved in citrate buffer (pH 6.0; Agilent Technologies
Inc.) for 20 min. Sections were incubated with primarymAbs for 1 h at room temperature, then treated using an
Envision+ kit (Agilent Technologies Inc.) for 30 min. Color
was developed using 3,3‑diaminobenzidine tetrahydrochloride (Agilent Technologies Inc.) for 2 min, and sections werethen counterstained with hematoxylin (FUJIFILM Wako Pure
Chemical Corporation).
Immunohistochemical analyses for frozen tissues. Histologic
sections (catalog no. T6235086‑1, BioChain Institute Inc.)
were incubated with 1 µg/ml of primary mAbs for 1 h atroom temperature and were then treated using an Envision+kit (Agilent Technologies Inc.) for 30 min. Color was developed using 3,3‑diaminobenzidine tetrahydrochloride (Agilent
Technologies Inc.) for 2 min, and sections were then counterstained with hematoxylin (FUJIFILM Wako Pure Chemical
Corporation).
Determination of the binding affinity. Cells were suspended in
100 µl serially diluted H2Mab‑19 (6 ng/ml‑100 µg/ml), followedby the addition of Alexa Fluor 488‑conjugated anti‑mouse IgG(1:200; Cell Signaling Technology, Inc.). Fluorescence microscopy data were collected using an EC800 Cell Analyzer(Sony Corp.). The dissociation constant (KD) was obtained byfitting binding isotherms to built‑in one‑site binding models in
GraphPad PRISM 6 (GraphPad Software, Inc.).
Antibody‑dependent cellular cytotoxicity. Six six‑week‑old
female BALB/c nude mice were purchased from Charles
River. After euthanization by cervical dislocation, spleens
were removed aseptically and single‑cell suspensions obtainedby forcing spleen tissues through a stainless steel mesh usinga syringe. Erythrocytes were lysed with a 10‑sec exposure toice‑cold distilled water. Splenocytes were washed with DMEM
and resuspended in DMEM with 10% FBS and used as effector
cells. Target cells were labeled with 10‑µg/ml Calcein AM(Thermo Fisher Scientific, Inc.) and resuspended in the same
medium. The target cells (2x10 4 cells/well) were plated in
96‑well plates and mixed with effector cells, anti‑HER2 antibodies, or control IgG (mouse IgG2b) (Sigma‑Aldrich Corp.).
After a 4‑h incubation, the Calcein AM release of supernatant from each well was measured. Fluorescence intensity
was determined using a microplate reader (Power Scan HT)(BioTek Instruments) with an excitation wavelength of 485 nm
and an emission wavelength of 538 nm. Cytolytic activity(as % of lysis) was calculated as: % lysis=(E‑S)/(M‑S) x100,

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where E is fluorescence of combined target and effector cells,
S is spontaneous fluorescence of target cells only, and M is
maximum fluorescence measured after lysing all cells witha buffer containing 0.5% Triton X‑100, 10 mM Tris‑HCl(pH 7.4), and 10 mM of EDTA.
Complement‑dependent cytotoxicity. Cells in DMEM supplemented with 10% FBS (2x104 cells/well) were plated in 96‑wellplates., and incubated for 5 h at 37̊C with either anti‑HER2
antibodies or control IgG (mouse IgG2b) (Sigma‑AldrichCorp.) and 10% of rabbit complement (Low‑Tox‑M Rabbit
Complement) (Cedarlane Laboratories). To assess cell
viability, an MTS [3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)​‑2‑(4‑sulfophenyl)‑2H‑tetrazolium; inner salt]
assay was performed using a CellTiter 96 AQueous assay kit(Promega).
Antitumor activity of H2Mab‑19 in the xenografts of breast
cancers. Sixteen six‑week‑old female BALB/c nude mice werepurchased from Charles River (Kanagawa, Japan) and used at
10 weeks of age. BT‑474 cells (0.3 ml of 1.33x108 cells/ml in
DMEM) were mixed with 0.5 ml BD Matrigel Matrix Growth
Factor Reduced (BD Biosciences). One hundred‑µl of thissuspension (5x106 cells) was injected subcutaneously into theleft flank. After day 1, 100 µg H2Mab‑19 and control mouse
IgG (Sigma‑Aldrich Corp.) in 100 µl PBS were injected i.p. into
treated and control mice, respectively. Additional antibodies
were then injected on days 7 and 14. Eighteen days after cell
implantation, all mice were euthanized by cervical dislocationand tumor diameters and volumes were determined as previously described (15).
Antitumor activity of H2Mab‑19 in xenografts of oral cancers.
Thirty‑two six‑week‑old female BALB/c nude mice werepurchased from Charles River and used at 10 weeks of age.
HSC‑2 or SAS cells in DMEM (0.3 ml with 1.33x108 cells/ml)
were mixed with 0.5 ml BD Matrigel Matrix Growth Factor
Reduced (BD Biosciences). A 100‑µl suspension containing
5x106 cells was injected subcutaneously into the left flank.
After day 1, 100 µg H 2 Mab‑19 and control mouse IgG(Sigma‑Aldrich Corp.) in 100 µl PBS were injected i.p. into
treated and control mice, respectively. Additional antibodies
were then injected on days 6 and 14. Twenty days after cell
implantation, all mice were euthanized by cervical dislocation.
Tumor diameters and volumes were determined as previously
described (15).
Statistical analyses. All data were expressed as mean ± SEM.
Statistical analysis used ANOVA and Tukey‑Kramer's testwith GraphPad Prism 6 (GraphPad Software, Inc.). P<0.05
was considered to indicate a statistically significant difference.
Results
Production of anti‑HER2 mAb. One mouse was immunizedwith the recombinant extracellular domain of HER2 (16),purified using the MAP tag system (17). Flow cytometry
was performed to check reactions with the LN229 cells(glioblastoma) and HER2‑overexpressing LN229 cells(LN229/HER2). LN229 cells endogenously express HER2

and some reaction with these cells was expected. The overexpression of HER2 in LN229/HERS2 cells would produce
a stronger reaction. One IgG2b subclass clone of H 2Mab‑19
was obtained, though almost all mAbs were in the mouse
IgG1 subclass. H 2Mab‑19 reacted with LN229/HER2 andweakly reacted with LN229 cells (Fig. 1A), indicating that
H2Mab‑19 is specific to HER2.
Characterization of H2Mab‑19. H2Mab‑19 recognized endogenous HER2 in a breast cancer cell line, BT‑474, which is
HER2‑positive (18), but did not react with a breast cancer cellline, MDA‑MB‑468, which is HER2‑negative (18) (Fig. 1A).
Further, H2Mab‑19 strongly reacted with endogenous HER2
in HO‑1‑u‑1 cells (oral cancer) and only weakly reacted withother oral cancer cell lines, Ca9‑22, HSC‑2, and SAS (Fig. 1A).
Using flow cytometry, binding affinities (KD) of H2Mab‑19 to
BT‑474, HSC‑2, and SAS cell lines were 2.3x10‑8, 9.5x10‑9 and5.5x10‑9 M, respectively. These results indicate that H2Mab‑19
maintains high affinity across HER2‑expressing cell lines.
H2Mab‑19 did not stain FFPE‑breast cancer tissues (Fig. S1).
In contrast, H 2Mab‑19 reacted with frozen breast cancer
tissues although the sensitivity of H2Mab‑77 was better thanthat of H2Mab‑19 (Fig. S2).
ADCC and CDC activities against breast and oral squamouscell carcinoma cell lines. This study examined whether
H 2Mab‑19 induced ADCC and CDC in HER2‑expressing
breast or OSCC cell lines. H 2Mab‑19 was a mouse IgG2b
subclass antibody that could possess both ADCC and CDC.
H 2Mab‑19 exhibited high ADCC activity against BT‑474,
HSC‑2, and SAS cells (Fig. 2A). High CDC activity was also
observed in BT‑474, HSC‑2, and SAS cells (Fig. 2B), suggestingthat H2Mab‑19 might exert antitumor activity in vivo.
Antitumor activity of H2Mab‑19 in mouse xenografts of breast
cancers. To study the antitumor activity of H2Mab‑19 on cell
growth in vivo, BT‑474 cells were implanted subcutaneously
in the flanks of nude mice. H 2Mab‑19 and control mouse
IgG were injected i.p. three times (days 1, 7, and 14 after cell
injection) into treated and control mice, respectively. Tumor
formation was observed in mice in both H 2Mab‑19‑treatedand control groups. H2Mab‑19 treatment significantly reduced
tumor development compared to development in control miceon days 5, 7, 12, 15, and 18 (Fig. 3A, upper). Weights of tumors
from H2Mab‑19‑treated mice were significantly less than for
tumors from IgG‑treated control mice (Fig. 3B, upper). BT‑474
xenografts on day 18 are shown in Fig. S3A. Resected tumors
are depicted in Fig. S3B. Total body weight was not significantly different between the two groups (Fig. S3C). We could
not show the histological data about the liver and kidney inthis study. HER2 was highly expressed in all cancer cells of
H2Mab‑19‑treated BT‑474 and control xenografts (Fig. S4).
Antitumor activities of H2Mab‑19 in the mouse xenografts oforal cancers. H2Mab‑19 possessed antitumor activity in mouse
xenografts of breast cancers. Whether this activity extended
to xenografts of oral cancers was also assessed. BT‑474 cells
expressed high levels of HER2 (Fig. 1A); HER2 levels were
however low in HSC‑2 and SAS cells (Fig. 1B). Nevertheless,
HSC‑2 and SAS are useful for investigation of antitumor

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849

Figure 1. Characterization of H2Mab‑19 using flow cytometry. (A) Glioblastoma cell lines (LN229, LN229/HER2), breast cancer cell lines (MDA‑MB‑468,
BT‑474), and oral cancer cell lines (Ca9‑22, HO‑1‑u‑1, HSC‑2, SAS) were treated with H2Mab‑19. Black line is negative control (PBS). (B) Determination ofbinding affinity of H2Mab‑19 for BT‑474, HSC‑2, and SAS using flow cytometry.

activity in vivo (16). Thus, HSC‑2 and SAS were used formouse xenografts of oral cancers.
Initially, HSC‑2 cells were implanted subcutaneously
into the flanks of nude mice. H 2Mab‑19 and mouse IgG
were injected i.p. three times (on days 1, 6, and 14 after cell
injections into treated and control mice, respectively. Tumor
formation was observed in mice in both groups. In comparison
to control mice, H2Mab‑19‑treated mice showed significantly
reduced tumor development on days 6, 10, 14, 17 and 20(Fig. 3A, middle). Weights of tumors from H2Mab‑19‑treatedmice were significantly less than for tumors from control
mice (Fig. 3B, middle). HSC‑2 xenograft mice are shownon day 20 in Fig. S5A and resected tumors are depicted inFig. S5B. Total body weights were not significantly different
between the two groups (Fig. S5C). We could not show thehistological data about the liver and kidney in this study.
HER2 was not expressed in cancer cells of H2Mab‑19‑treatedor control groups (Fig. S6). HER2 expression was diminishedin HSC‑2 xenografts, and H 2Mab‑19 did not exert effective
antitumor activity.
For the second xenograft model of oral cancers, SAS cells
were subcutaneously implanted into the flanks of nude mice.
H2Mab‑19 and mouse IgG were injected i.p. thrice, on days 1, 6,

and 14 after cell injections into the mice, into treated and control
mice, respectively. Tumor formation was observed in mice inboth treated and control groups. In comparison to IgG‑treatedcontrol mice, H2Mab‑19 significantly reduced tumor development on days 14, 17, and 20 (Fig. 3A, lower). Weights of tumors
from H 2Mab‑19‑treated mice were significantly less thantumors from IgG‑treated control Mice (Fig. 3B, lower). The
SAS xenografts on day 20 are shown in Fig. S7A and resected
tumors are depicted in Fig. S7B. Total body weights were notsignificantly different between the two groups (Fig. S7C).
We could not show the histological data about the liver andkidney in this study. HER2 was not expressed in cancer cells of
H2Mab‑19‑treated and control groups (Fig. S8). HER2 expression was diminished in SAS xenografts, and H2Mab‑19 did notexert effective antitumor activity.
Discussion
Using CasMab technology (19), several anti‑HER2 mAbs,
including H2Mab‑77 (14), H2Mab‑119 (20), and H2Mab‑139 (16)were identified. These antibodies are useful for flow cytometry, western blot, and immunohistochemical analyses.
Because the subclass of these mAbs is mouse IgG1, they do not

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Figure 2. ADCC and CDC activity. (A) ADCC activity against BT474, HSC‑2 and SAS cells. (B) CDC activity against BT474, HSC‑2 and SAS cells. **P<0.01;*
P<0.05; n.s., not significant; ADCC, antibody‑dependent cellular cytotoxicity; CDC, complement‑dependent cytotoxicity.

possess antibody‑dependent cellular cytotoxicity (ADCC) orcomplement‑dependent cytotoxicity (CDC).
The first objective of this study was the development ofan anti‑HER2 mAb in either IgG2a or IgG2b subclasses using
CasMab technology. Both IgG2a (21) and IgG2b antibodies (22)show ADCC and CDC activity. The second objective was toinvestigate anti‑HER2 activity using oral cancer cell lines;
anti‑HER2 mAbs have not been investigated for their activity
against oral cancers. The first objective was met throughisolation of H2Mab‑19 from the IgG2b subclass (Fig. 1). This
antibody could then be used to investigate ADCC and CDC
activity in vitro and antitumor activity in vivo. H 2Mab‑19
showed both ADCC and CDC activity against breast ororal cancer cell lines (Fig. 2). Further, H 2Mab‑19 exerted
antitumor activity against both breast cancer and oral cancer

xenografts (Fig. 3). These results demonstrated two important issues: i) anti‑HER2 mAbs from the IgG2b subclass
could be developed using our original CasMab technology,
and ii) anti‑HER2 mAbs from IgG2b subclass could possess
ADCC, CDC, and antitumor activities. Recently, Fiedler et alreported that TrasGEX, an ADCC‑enhanced version of
trastuzumab, showed antitumor activity in 50% of evaluatedpatients from a phase Ⅰ study (23). They showed that TrasGEX
exhibited similar pharmacokinetics to those of trastuzumab
and was safe and well‑tolerated by patients with solid tumors.
These data are consistent with the designation of HER2
as a promising target for the treatment of HER2‑amplified
tumors. Trastuzumab and TrasGEX are known as beneficial
anti‑HER2 mAbs for targeting breast or stomach cancers,
H2Mab‑19 could be also a useful tool for investigating ADCC,

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851

Figure 3. Evaluation of antitumor activity of H2Mab‑19. (A) Tumor volume and (B) tumor weight measured from BT‑474 (upper), HSC‑2 (middle) and SAS(lower) xenografts. Values are mean ± SEM. **P<0.01; *P<0.05.

CDC, antitumor activities for oral cancers. Further investigation of the mechanism of antitumor activity by H2Mab‑19, andthe development of antibody‑engineered antibodies, including
chimeric or humanized H2Mab‑19 or its single chain (sc) Fv,
are aims for future studies.
Oral cancer accounts for approximately 2% of all cancer
cases worldwide (24). Annually, more than 350,000 individuals are diagnosed with oral cancer and these diseases
prove fatal for 170,000 of these people. Major risk factors fororal cancer are the use of tobacco and alcohol (25). Decreasedsmoking and drinking has resulted in a decline in the incidence of oral cancer. However, recent studies have reported anincrease in the number of young patients diagnosed with these
diseases (26,27).
More than 50% of oral cancers occur in tongue tissue
and on the floor of the mouth. Other locations include thebuccal mucosa, gingiva, lip and palate (28). HER2 expression was assessed in four oral cancer cell lines of different
origin, including Ca9‑22 (gingiva), HO‑1‑u‑1 (mouth floor),

HSC‑2 (oral cavity), and SAS (tongue). HER2 expression wasobserved in all cell lines (Fig. 1), indicating that expression is
independent of location in the oral cavity.
Oral cancers display several histological tumor types,
including squamous cell carcinoma (SCC), adenocarcinoma,
mucoepidermoid carcinoma, adeno cystic carcinoma andosteosarcoma. SCC is most common, accounting for over 90%
of all disease (29). Treatment of oral SCC (OSCC) depends forthe most part on stage. Early stages (stage‑Ⅰ and ‑Ⅱ) are treated
via surgery or radiotherapy (RT) alone. Advanced stages(stage‑Ⅲ and ‑Ⅳ) require a combination of surgery, RT and
chemotherapy (CT) (30). Cisplatin (CDDP) is mainly used for
CT of OSCCs, often combined with 5‑fluorouracil (5‑FU) anddocetaxel (31,32). Other anticancer agents such as carboplatin,
paclitaxel, and methotrexate (MTX) can be useful (33), but
useful drugs with specific molecular targets are limited.
Cetuximab, a mouse‑human chimeric antibody (IgG1) thattargets epidermal growth factor receptor (EGFR), was recently
approved for treatment of oral cancer. Several studies report its

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EXPERIMENTAL AND THERAPEUTIC MEDICINE 20: 846-853, 2020

effectiveness against locoregionally advanced head and neckcancer and recurrent or metastatic squamous cell carcinoma of
the head and neck (34‑36). Advances in diagnosis and therapeutic techniques have improved the overall 5‑year survival
rate to 70%. However, the 5‑year survival rate in stage Ⅳ is
only 40% (37) and further treatments need to be developed.
In this study, HER2 is shown to be expressed in oral cancers,
and anti‑HER2 mAbs have useful for antitumor activity. Thus,anti‑HER2 therapies using trastuzumab could be valuablefor oral cancer treatment. Immunohistochemically, HER2
expressed was reported in only 1.4% (38) of oral cancer,
though it is expressed in 10.4% of breast cancers (39). Thus,targeting only HER2 may not be sufficient for treating oral
cancers. Despite the low HER2 overexpression/amplification
rate of only 1‑2%, those few patients may possibly benefit fromanti‑HER2 therapy because an antitumor effect of combined
gefitinib and trastuzumab or cetuximab and trastuzumab treatment on HNSCC in vitro were demonstrated (40,41). Pursuing
multiple targets, such as EGFR and HER2, may be needed for
effective therapy.
Acknowledgements
The authors would like to thank Ms. Akiko Harakawa(Institute of Microbial Chemistry (BIKAKEN), Numazu,
Microbial Chemistry Research Foundation) for technicalassistance of animal experiments, and Mr. Takuro Nakamura,
Ms. Miyuki Yanaka, Ms. Saori Handa, Ms. Saki Okamoto, andMr. Yu Komatsu (Department of Antibody Drug Development,
Tohoku University Graduate School of Medicine) for technicalassistance of in vitro experiments.
Funding
This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under (grantnos. JP19am0401013, JP19am0101078 and JP19ae0101028),
and by Japan Society for the Promotion of Science (JSPS)
Grants‑in‑Aid for Scientific Research (KAKENHI; grantnos. 17K07299 and 19K07705).
Availability of data and materials
The datasets used and/or analyzed during the study areavailable from the corresponding author on reasonable request.
Authors' contributions
JT and TO performed experiments. MKK analyzed experimental data. MK, HH, and YK designed the current study andwrote the manuscript.
Ethics approval and consent to participate
Animal experiments described in the hybridoma production
were approved by the Animal Care and Use Committee of
Tohoku University (permit no. 2016MdA‑153). Animal studies
for ADCC were approved by the institutional committee forexperiments of the Institute of Microbial Chemistry (permit
no. 2019‑066). Animal studies for the antitumor activity wereapproved by the institutional committee for experiments of theInstitute of Microbial Chemistry (permit no. 2019‑014).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References
1. Howlader N, Altekruse SF, Li CI, Chen VW, Clarke CA, Ries LA
and Cronin KA: US incidence of breast cancer subtypes definedby joint hormone receptor and HER2 status. J Natl Cancer
Inst 106: dju055, 2014.
2. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A and
McGuire WL: Human breast cancer: Correlation of relapse
and survival with amplification of the HER‑2/neu oncogene.
Science 235: 177‑182, 1987.
3. Xie S, Zhang H, Wang X, Ge Q and Hu J: The relative efficacy andsafety of targeted agents used in combination with chemotherapy
in treating patients with untreated advanced gastric cancer:
A network meta‑analysis. Oncotarget 8: 26959‑26968, 2017.
4. Harder J, Ihorst G, Heinemann V, Hofheinz R, Moehler M,
Buechler P, Kloeppel G, Röcken C, Bitzer M, Boeck S, et al:Multicentre phase II trial of trastuzumab and capecitabine inpatients with HER2 overexpressing metastatic pancreatic cancer.
Br J Cancer 106: 1033‑1038, 2012.
5. Kim EK, Kim KA, Lee CY and Shim HS: The frequency andclinical impact of HER2 alterations in lung adenocarcinoma.
PLoS One 12: e0171280, 2017.
6. Seo AN, Kwak Y, Kim DW, Kang SB, Choe G, Kim WH and
Lee HS: HER2 status in colorectal cancer: Its clinical significance and the relationship between HER2 gene amplification andexpression. PLoS One 9: e98528, 2014.
7. Lambert JM and Chari RV: Ado‑trastuzumab emtansine(T‑DM1): An antibody‑drug conjugate (ADC) for HER2‑positive
breast cancer. J Med Chem 57: 6949‑6964, 2014.
8. Valabrega G, Montemurro F and Aglietta M: Trastuzumab:
Mechanism of action, resistance and future perspectives in
HER2‑overexpressing breast cancer. Ann Oncol 18: 977‑984,
2007.
9. Amiri‑Kordestani L, Wedam S, Zhang L, Tang S, Tilley A,
Ibrahim A, Justice R, Pazdur R and Cortazar P: First FDA
approval of neoadjuvant therapy for breast cancer: Pertuzumab
for the treatment of patients with HER2‑positive breast cancer.
Clin Cancer Res 20: 5359‑5364, 2014.
10. Slamon DJ, Leyland‑Jones B, Shak S, Fuchs H, Paton V,
Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, et al:Use of chemotherapy plus a monoclonal antibody against HER2
for metastatic breast cancer that overexpresses HER2. N Engl J
Med 344: 783‑792, 2001.
11. Swain SM, Kim SB, Cortés J, Ro J, Semiglazov V, Campone M,
Ciruelos E, Ferrero JM, Schneeweiss A, Knott A, 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 14: 461‑471, 2013.
12. Doi T, Shitara K, Naito Y, Shimomura A, Fujiwara Y, Yonemori K,
Shimizu C, Shimoi T, Kuboki Y, Matsubara N, et al: Safety,
pharmacokinetics, and antitumour activity of trastuzumab deruxtecan (DS‑8201), a HER2‑targeting antibody‑drug conjugate, inpatients with advanced breast and gastric or gastro‑oesophageal
tumours: A phase 1 dose‑escalation study. Lancet Oncol 18:1512‑1522, 2017.
13. Nakada T, Sugihara K, Jikoh T, Abe Y and 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) 67: 173‑185, 2019.
14. Itai S, Fujii Y, Kaneko MK, Yamada S, Nakamura T, Yanaka M,
Saidoh N, Chang YW, Handa S, Takahashi M, et al: H2Mab‑77 isa sensitive and specific anti‑HER2 monoclonal antibody against
breast cancer. Monoclon Antib Immunodiagn Immunother 36:
143‑148, 2017.

TAKEI et al: AN ANTI-HER2 mAb FOR ORAL CANCERS

15. Kato Y, Kunita A, Abe S, Ogasawara S, Fujii Y, Oki H,
Fukayama M, Nishioka Y and Kaneko MK: The chimericantibody chLpMab‑7 targeting human podoplanin suppresses
pulmonary metastasis via ADCC and CDC rather than via its
neutralizing activity. Oncotarget 6: 36003‑36018, 2015.
16. Kaneko MK, Yamada S, Itai S and Kato Y: Development ofan anti‑HER2 monoclonal antibody H2Mab‑139 against coloncancer. Monoclon Antib Immunodiagn Immunother 37: 59‑62,2018.
17. Fujii Y, Kaneko MK and Kato Y: MAP tag: A novel tagging
system for protein purification and detection. Monoclon AntibImmunodiagn Immunother 35: 293‑299, 2016.
18. Al‑Saden N, Lam K, Chan C and Reilly RM: Positron‑emission
tomography of HER2‑positive breast cancer xenografts inmice with 89Zr‑labeled trastuzumab‑DM1: A comparison with89
Zr‑labeled trastuzumab. Mol Pharm 15: 3383‑3393, 2018.
19. Kato Y and Kaneko MK: A cancer‑specific monoclonal antibody
recognizes the aberrantly glycosylated podoplanin. Sci Rep 4:5924, 2014.
20. Yamada S, Itai S, Nakamura T, Chang YW, Harada H, Suzuki H,
Kaneko MK and Kato Y: Establishment of H 2Mab‑119, ananti‑human epidermal growth factor receptor 2 monoclonal antibody, against pancreatic cancer. Monoclon Antib Immunodiagn
Immunother 36: 287‑290, 2017.
21. Kaneko MK, Nakamura T, Honma R, Ogasawara S, Fujii Y,
Abe S, Takagi M, Harada H, Suzuki H, Nishioka Y and
Kato Y: Development and characterization of anti‑glycopeptide
monoclonal antibodies against human podoplanin, using
glycan‑deficient cell lines generated by CRISPR/Cas9 and
TALEN. Cancer Med 6: 382‑396, 2017.
22. Ogasawara S, Kaneko MK and Kato Y: LpMab‑19 recognizes
sialylated O‑Glycan on Thr76 of human podoplanin. Monoclon
Antib Immunodiagn Immunother 35: 245‑253, 2016.
23. Fiedler W, Stoeger H, Perotti A, Gastl G, Weidmann J,
Dietrich B, Baumeister H, Danielczyk A, Goletz S, Salzberg M
and De Dosso S: Phase I study of TrasGEX, a glyco‑optimised
anti‑HER2 monoclonal antibody, in patients with HER2‑positive
solid tumours. ESMO Open 3: e000381, 2018.
24. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA and
Jemal A: Global cancer statistics 2018: GLOBOCAN estimatesof incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68: 394‑424, 2018.
25. Hashibe M, Brennan P, Chuang SC, Boccia S, Castellsague X,
Chen C, Curado MP, Dal Maso L, Daudt AW, Fabianova E, et al:Interaction between tobacco and alcohol use and the risk ofhead and neck cancer: Pooled analysis in the international headand neck cancer epidemiology consortium. Cancer Epidemiol
Biomarkers Prev 18: 541‑550, 2009.
26. Tota JE, Anderson WF, Coffey C, Califano J, Cozen W, Ferris RL,
St John M, Cohen EEW and Chaturvedi AK: Rising incidence oforal tongue cancer among white men and women in the UnitedStates, 1973‑2012. Oral Oncol 67: 146‑152, 2017.
27. Hussein AA, Helder MN, de Visscher JG, Leemans CR,
Braakhuis BJ, de Vet HCW and Forouzanfar T: Global incidenceof oral and oropharynx cancer in patients younger than 45 years
versus older patients: A systematic review. Eur J Cancer 82:
115‑127, 2017.

853

28. Bagan J, Sarrion G and Jimenez Y: Oral cancer: Clinical features.
Oral Oncol 46: 414‑417, 2010.
29. Rivera C: Essentials of oral cancer. Int J Clin Exp Pathol 8:11884‑11894, 2015.
30. Guneri P and Epstein JB: Late stage diagnosis of oral cancer:
Components and possible solutions. Oral Oncol 50: 1131‑1136,
2014.
31. Vokes EE: Induction chemotherapy for head and neck cancer:
Recent data. Oncologist 15 (Suppl 3): S3‑S7, 2010.
32. Marcazzan S, Varoni EM, Blanco E, Lodi G and Ferrari M:
Nanomedicine, an emerging therapeutic strategy for oral cancer
therapy. Oral Oncol 76: 1‑7, 2018.
33. Furness S, Glenny AM, Worthington HV, Pavitt S, Oliver R,
Clarkson JE, Macluskey M, Chan KK and Conway DI: Interventions
for the treatment of oral cavity and oropharyngeal cancer:
Chemotherapy. Cochrane Database Syst Rev CD006386: 2011.
34. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB,
Jones CU, Sur R, Raben D, Jassem J, et al: Radiotherapy plus
cetuximab for squamous‑cell carcinoma of the head and neck.
N Engl J Med 354: 567‑578, 2006.
35. Vermorken JB, Mesia R, Rivera F, Remenar E, Kawecki A,
Rottey S, Erfan J, Zabolotnyy D, Kienzer HR, Cupissol D, et al:
Platinum‑based chemotherapy plus cetuximab in head and neckcancer. N Engl J Med 359: 1116‑1127, 2008.
36. Naruse T, Yanamoto S, Matsushita Y, Sakamoto Y, Morishita K,
Ohba S, Shiraishi T, Yamada SI, Asahina I and Umeda M:
Cetuximab for the treatment of locally advanced and recurrent/metastatic oral cancer: An investigation of distant metastasis.
Mol Clin Oncol 5: 246‑252, 2016.
37. Amit M, Yen TC, Liao CT, Chaturvedi P, Agarwal JP,
Kowalski LP, Ebrahimi A, Clark JR, Kreppel M, Zöller J, et al:
Improvement in survival of patients with oral cavity squamouscell carcinoma: An international collaborative study. Cancer 119:4242‑4248, 2013.
38. Hanken H, Gaudin R, Gröbe A, Fraederich M, Eichhorn W,
Smeets R, Simon R, Sauter G, Grupp K, Izbicki JR, et al:
Her2 expression and gene amplification is rarely detectablein patients with oral squamous cell carcinomas. J Oral Pathol
Med 43: 304‑308, 2014.
39. Yan M, Schwaederle M, Arguello D, Millis SZ, Gatalica Z and
Kurzrock R: HER2 expression status in diverse cancers: Review
of results from 37,992 patients. Cancer Metastasis Rev 34:
157‑164, 2015.
40. Kondo N, Ishiguro Y, Kimura M, Sano D, Fujita K, Sakakibara A,
Taguchi T, Toth G, Matsuda H and Tsukuda M: Antitumor effectof gefitinib on head and neck squamous cell carcinoma enhanced
by trastuzumab. Oncol Rep 20: 373‑378, 2008.
41. Kondo N, Tsukuda M, Sakakibara A, Takahashi H, Hyakusoku H,
Komatsu M, Niho T, Nakazaki K and Toth G: Combined
molecular targeted drug therapy for EGFR and HER‑2 in headand neck squamous cell carcinoma cell lines. Int J Oncol 40:
1805‑1812, 2012.
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