INTERNATIONAL JOURNAL OF MOlecular medicine 46: 1443-1452, 2020

Anti‑EGFR monoclonal antibody 134‑mG2a exerts antitumor
effects in mouse xenograft models of oral squamous cell carcinoma
HIDEKI HOSONO1*, JUNKO TAKEI1,2*, TOMOKAZU OHISHI3*, MASATO SANO1, TEIZO ASANO1,YUSUKE SAYAMA1, TAKURO NAKAMURA1, MIYUKI YANAKA1, MANABU KAWADA3,HIROYUKI HARADA2, MIKA KATO KANEKO1 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),
Microbial Chemistry Research Foundation, Numazu‑shi, Shizuoka 410‑0301;
4
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi 980‑8575, Japan
Received June 1, 2020; Accepted July 22, 2020
DOI: 10.3892/ijmm.2020.4700
Abstract. The epidermal growth factor receptor (EGFR), atransmembrane receptor and member of the human epidermal
growth factor receptor (HER) family of receptor tyrosine
kinases, is a critical mediator of cell growth and differentiation. EGFR forms homo‑ or heterodimers with other HER
family members to activate downstream signaling cascades
in a number of cancer cells. In a previous study, the authors
established an anti‑EGFR monoclonal antibody (mAb),
EMab‑134, by immunizing mice with the ectodomain ofhuman EGFR. EMab‑134 binds specifically to endogenous
EGFR and can be used to detect receptor on oral cancer

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*

Contributed equally

Abbreviations: ADCC, antibody‑dependent cellular cytotoxicity;
ATCC, American Type Culture Collection; CasMab, cancer‑specificmonoclonal antibody; CDC, complement‑dependent cytotoxicity;
CHO, Chinese hamster ovary; DAB, 3,3'‑diaminobenzidine
tetrahydrochloride; DMEM, Dulbecco's modified Eagle's medium;
EDTA, ethylenediaminetetraacetic acid; EGFR, epidermal growth
factor receptor; FBS, fetal bovine serum; JCRB, Japanese Collectionof Research Bioresources Cell Bank; HER, human epidermal growth
factor receptor; mAb, monoclonal antibody; n.s., not significant;
OSCC, oral squamous cell carcinoma; PBS, phosphate‑bufferedsaline; PDPN, podoplanin; PODXL, podocalyxin; RPMI, Roswell
Park Memorial Institute; PVDF, polyvinylidene difluoride; SEM,
standard error of the mean
Key words: EGFR, monoclonal antibody, ADCC, CDC, antitumor
activity, oral cancer

cell lines by flow cytometry and western blot analysis; thisantibody is also effective for the immunohistochemical evaluation of oral cancer tissues. In the present study, the subclassof EMab‑134 was converted from IgG1 to IgG2a (134‑mG2a)
to facilitate antibody‑dependent cellular cytotoxicity (ADCC)
and complement‑dependent cytotoxicity (CDC). The dissociation constants (K Ds) of EMab‑134 and 134‑mG2a against
EGFR‑expressing CHO‑K1 (CHO/EGFR) cells were determined by flow cytometry to be 3.2x10 ‑9 M and 2.1x10 ‑9 M,
respectively; these results indicate that 134‑mG2a has a higher
binding affinity than EMab‑134. The 134‑mG2a antibody was
more sensitive than EMab‑134 with respect to antigen detection in oral cancer cells in both western blot analysis andimmunohistochemistry applications. Analysis in vitro revealed
that 134‑mG2a contributed to high levels of ADCC and CDC
in experiments targeting CHO/EGFR, HSC‑2, and SAS cells.
Moreover, the in vivo administration of 134‑mG2a significantly
inhibited the development of CHO/EGFR, HSC‑2, and SAS
mouse xenografts in comparison to the results observed in
response to EMab‑134. Taken together, the findings of thepresent study demonstrate that the newly‑formulated 134‑mG2a
is useful for detecting EGFR by flow cytometry, western
blot analysis and immunohistochemistry. Furthermore, thein vivo results suggested that it may also be useful as part ofa therapeutic regimen for patients with EGFR‑expressing oral
cancer.
IntroductionMore than 350,000 individuals are diagnosed with oral cancer
annually, and oral cancer will ultimately prove fatal in almost
half of those diagnosed with the disease (1). Of the defined
histological types of oral cancer, >90% of patients are diagnosed with oral squamous cell carcinoma (OSCC), which
typically arises on the lips or within the oral cavity (2). The
most effective treatments currently available for OSCC dependon its clinical stage at presentation. Although stage‑I and ‑II
OSCCs are treated with surgery or radiotherapy, advanced

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HOSONO et al: ANTITUMOR ACTIVITIES OF AN ANTI-EGFR mAb

stage‑III and ‑IV disease is treated with a combination ofsurgery, radiotherapy and chemotherapy (3). Chemotherapeutic
regimens typically include cisplatin as a first‑line agent; it
is often combined with docetaxel or 5‑fluorouracil (4,5).
Paclitaxel, methotrexate and carboplatin can be also used in
the treatment of OSCCs (6); however, there is only limited
information available on the efficacy of molecular targeting
drugs and/or antibody‑based therapies for OSCC.
The epidermal growth factor receptor (EGFR) is a memberof the human epidermal growth factor receptor (HER) familyof receptor tyrosine kinases, and is involved in cell growthand differentiation (7‑9). EGFR forms homo‑ or heterodimers
with other HER family members, such as HER2 and HER3,
and thereby activate downstream signaling cascades. Thesepathways are frequently dysregulated in malignant diseases,
including OSCC, often via the overexpression of EGFR (10).
Nimotuzumab is a humanized monoclonal antibody (mAb)directed against the extracellular domain of the EGFR thathas been shown to have clinical efficacy in various types ofcancer (11). Although nimotuzumab has been approved in
29 countries for use in the treatment of advanced head andneck carcinoma, esophageal cancer, nasopharyngeal carcinoma and pancreatic cancer, only modest success has beenachieved with respect to the treatment of recurrent and/or
metastatic OSCC (12). Although a number of EGFR‑targeted
therapies have been used in patients with OSCC, treatment
failures due to the low response rates and acquired resistancehave been reported (13).
In a previous study by the authors, mice were immunized with purified recombinant EGFR, and successfully
produced monoclonal EMab‑134 (mouse IgG1, kappa). This
antibody detected endogenous EGFR in oral cancers inapplications including flow cytometry, western blot analysis
and immunohistochemistry (14). For example, when usedin immunohistochemical analysis, EMab‑134 reacted with
its target antigen in 36 of 38 (94.7%) oral cancer specimens.
The minimum epitope of EMab‑134 was determined to be
377‑ RGDSFTHTPP‑386 (15). Although EMab‑134 has provento be very useful for the detection of EGFR, the mouse IgG1
subclass does not facilitate antibody‑dependent cellularcytotoxicity (ADCC) or complement‑dependent cytotoxicity(CDC) activities.
To address this issue, in the present study, EMab‑134 (IgG1
subclass) was converted into 134‑mG2a of the mouse IgG2a
subclass. It was then determined whether 134‑mG2a exhibits
ADCC, CDC, and in vivo antitumor activities against OSCCs.
Materials and methods
Antibodies. Anti‑EGFR mAb EMab‑134 (mouse IgG1,
kappa) was developed as previously described (14). To
generate 134‑mG2a, V H cDNA of EMab‑134 and CH mouse
IgG2a were subcloned into pCAG‑Ble vector, and V L and
C L cDNAs of EMab‑134 were subcloned into pCAG‑Neo
vector (FUJIFILM Wako Pure Chemical Corporation),
respectively. Vectors were transfected into ExpiCHO‑S cells
using the ExpiCHO Expression System (Thermo Fisher
Scientific, Inc.). The resulting mAb, 134‑mG2a, was purified
with Protein G‑Sepharose (GE Healthcare Bio‑Sciences).
Mouse IgG (cat. no. I8765), IgG1 (cat. no. M7894), andIgG2a (cat. no. M7769) were purchased from Sigma‑Aldrich;
Merck KGaA.
Cell lines. The CHO‑K1 cell line was obtained from theAmerican Type Culture Collection (ATCC). Human EGFR‑​
expressing CHO‑K1 cells (CHO/EGFR) were previously
established by the transfection of pCAG/PA‑EGFR‑RAP‑MAP
into CHO‑K1 cells using Lipofectamine LTX (Thermo Fisher
Scientific, Inc.) (16). The amino acid sequences of each tag wereas follows: PA tag (17), 12 amino acids (GVAMPGAEDDVV);RAP tag (18), 12 amino acids (DMVNPGLEDRIE); andMAP tag (19), 12 amino acids (GDGMVPPGIEDK). OSCC
cell lines, including HSC‑2 (oral cavity) and SAS (tongue)
were obtained from the Japanese Collection of Research
Bioresources Cell Bank (JCRB). CHO‑K1 and CHO/EGFR
were cultured in Roswell Park Memorial Institute (RPMI)‑1640
medium (Nacalai Tesque, Inc.). The HSC‑2 and SAS cells were
cultured in Dulbecco's modified Eagle's medium (DMEM;
Nacalai Tesque, Inc.). Cell culture medium was supplemented
with 10% heat‑inactivated fetal bovine serum (FBS; Thermo
Fisher Scientific, Inc.), 100 units/ml of penicillin, 100 µg/ml
streptomycin, and 0.25 µg/ml amphotericin B (Nacalai Tesque,Inc.). Cells were cultured at 37˚C in a humidified 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 ADCC and antitumor activity were approved by theInstitutional Committee for Experiments of the Institute ofMicrobial Chemistry (Permit. no. 2019‑049 for ADCC assays,
2019‑046 for antitumor experiments). Mice were maintainedin a pathogen‑free environment (23±2˚C, 55±5% humidity)
on an 11‑h light/13‑h dark cycle with food and water supplied
ad libitum across the experimental period. Mice were monitored for health and weight every 2 or 5 days during the 3‑weekperiod of each experiment. The loss of original body weight
to a point >25% and/or a maximum tumor size >3,000 mm 3
were identified as humane endpoints for euthanasia. Mice
were euthanized by cervical dislocation; death was verified byrespiratory and cardiac arrest.
Flow cytometry. Cells were harvested by brief exposure
to 0.25% trypsin/1 mM ethylenediamine tetra acetic acid
(EDTA, Nacalai Tesque, Inc.). After washing with 0.1% bovine
serum albumin in phosphate‑buffered saline (PBS), cells weretreated with 1 µg/ml of anti‑EGFR mAbs for 30 min at 4˚Cfollowed by Alexa Fluor 488‑conjugated anti‑mouse IgG at adilution of 1:1,000 (cat. no. 4408S; Cell Signaling Technology,Inc.) for 30 min at 4˚C. Fluorescence data were collected using
an SA3800 Cell Analyzer (Sony Corp.).
Western blot analyses. Cell pellets were suspended using lysis
buffer (1% Triton X‑100 and 50 µg/ml aprotinin in PBS) on
ice for 15 min. Following centrifugation (20,630 x g, 15 min,
4˚C), cell lysates were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.). The samples were electrophoresed on 5‑20% polyacrylamide gels (Nacalai Tesque,Inc.) and transferred onto polyvinylidene difluoride (PVDF)
membranes (Merck KGaA). After blocking with 4% skimINTERNATIONAL JOURNAL OF MOlecular medicine 46: 1443-1452, 2020

milk (Nacalai Tesque, Inc.) for 1 h, the membranes wereincubated with anti‑EGFR mAbs or anti‑β‑actin (1 µg/ml) for
1 h, followed by incubation with HRP‑conjugated anti‑mouse
immunoglobulins at a 1:2,000 dilution (Agilent Technologies,Inc.) for 1 h. The membranes were developed with the
ImmunoStar LD Chemiluminescence Reagent (FUJIFILM
Wako Pure Chemical Corporation) using the Sayaca‑Imager(DRC Co., Ltd.). All western blot analysis procedures were
performed at room temperature.
Immunohistochemical analyses. Histological sections(4‑µm‑thick) of an oral cancer tissue array (cat. no. OR601c;
US Biomax, Inc.) were directly autoclaved in EnVision FLEX
Target Retrieval Solution High pH (Agilent Technologies, Inc.)for 20 min. The sections were then incubated with 5 µg/ml
anti‑EGFR mAbs for 1 h at room temperature and treatedusing an Envision+ kit (Agilent Technologies, Inc.) for 30 min.
Color was developed using 3,3'‑diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2 min,and the sections were then counterstained with hematoxylin(FUJIFILM Wako Pure Chemical Corporation). Hematoxylin
and eosin (H&E) staining was performed using consecutive
tissue sections as follows: Hematoxylin staining (FUJIFILM
Wako Pure Chemical Corporation) for 5 min and eosin staining(FUJIFILM Wako Pure Chemical Corporation) for 2 minat room temperature. Leica DMD108 (Leica MicrosystemsGmbH) was used to examine the sections and obtain images.
Determination of the binding affinity. The cells weresuspended in 100 µl of serially diluted anti‑EGFR mAbs
(0.6‑10 µg/ml) followed by the addition of Alexa Fluor
488‑conjugated anti‑mouse IgG (1:1,000; Cell Signaling
Technology, Inc.). Fluorescence data were collected using an
EC800 Cell Analyzer (Sony Corp.). The dissociation constant(K D) was calculated by fitting binding isotherms to built‑in
one‑site binding models in GraphPad Prism 7 (GraphPad
Software, Inc.).
ADCC. A total of 6 female 6‑week‑old BALB/c nude mice
(weighing 15‑18 g) were purchased from Charles River
Laboratories, Inc. Spleen cells from 6 mice were used as thesource of natural killer (NK) cells for the evaluation of ADCC,
which has been reported previously (20). Following euthanasia
by cervical dislocation, the spleens were removed asepticallyand single‑cell suspensions were obtained by forcing spleen
tissues through a sterile cell strainer (352360, BD Falcon,
Corning, Inc.) using a syringe. Erythrocytes were lysed witha 10‑sec exposure to ice‑cold distilled water. Splenocytes
were washed with DMEM and resuspended in DMEM with
10% FBS; this preparation was used as effector cells. Target
tumor cells were labeled with 10 µg/ml Calcein AM (Thermo
Fisher Scientific, Inc.) and resuspended in the same medium.
The target cells (2x104 cells/well) were plated in 96‑well plates
and mixed with effector cells (effector/target cell ratio, 50),
100 µg/ml of anti‑EGFR antibodies or control IgGs. Following
a 4‑h incubation at 37˚C, the release of Calcein AM into thesupernatant was measured in each well. The fluorescenceintensity was determined using a microplate reader (Power
Scan HT; BioTek Instruments, Inc.) with an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

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Cytolytic activity (% lysis) was calculated using the equation% lysis=(E‑S)/(M‑S) x100, where ‘E’ is the fluorescencemeasured in combined cultures of target and effector cells, ‘S’is the spontaneous fluorescence of target cells only, and ‘M’is the maximum fluorescence measured following the lysis of
all cells with a buffer containing 0.5% Triton X‑100, 10 mM
Tris‑HCl (pH 7.4) and 10 mM of EDTA.
CDC. The cells (2x10 4 cells/well) were plated in 96‑wellplates and mixed with rabbit complement (final dilution 1:10;
Low‑Tox‑M Rabbit Complement; Cedarlane Laboratories)
together with 100 µg/ml of anti‑EGFR or control IgGs.
Following 5 h incubation at 37˚C, 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 Corp.).
Antitumor activity of 134‑mG2a in xenografts of CHO/EGFR
cells. A total of 24 female BALB/c nude mice (5 weeks
old, weighing 14‑17 g) were purchased from Charles River
Laboratories, Inc. and used in experiments once they reached
7 weeks of age. CHO/EGFR 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); 100 µl of this suspension(5x106 cells) was injected subcutaneously into the left flanksof the mice. On day 1 post‑inoculation, 100 µg of EMab‑134(n=8), 134‑mG2a (n=8), or control mouse IgG (n=8) in 100 µl
PBS were injected intraperitoneally. Additional antibody
inoculations were performed on days 7 and 14. At 21 days
following cell implantation, all mice were euthanized bycervical dislocation; tumor diameters and volumes were
determined as previously described (21).
Antitumor activity of 134‑mG2a in xenografts of oral cancers.
A total of 48 female BALB/c nude mice (5 weeks old, weighing
14‑17 g) were purchased from Charles River Laboratories,
Inc. and used in experiments once they reached 7 weeks ofage. The HSC‑2 and SAS 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); 100 µl of this suspension(5x106 cells) was injected subcutaneously into the left flanks ofthe mice. On day 1 post‑inoculation, 100 µg of EMab‑134 (n=8in each group), 134‑mG2a (n=8 in each group), or control mouse
IgG (n=8 in each group) in 100 µl PBS were injected intraperitoneally. Additional antibody inoculations were performed ondays 7 and 14. At 18 days following cell implantation, all mice
were euthanized by cervical dislocation, and tumor diametersand volumes were determined.
Statistical analyses. All data are expressed as themeans ± standard error of the mean (SEM). Statisticalanalysis was performed using one‑way ANOVA followed
by Tukey‑Kramer's test using R statistical (R Foundation for
Statistical Computing). A value of P<0.05 was adopted as alevel of statistical significance.
Results
Generation and characterization of 134‑mG 2a , a mouse
IgG2a‑type anti‑EGFR antibody. As mouse IgG2a subclass1446

HOSONO et al: ANTITUMOR ACTIVITIES OF AN ANTI-EGFR mAb

facilitates both ADCC and CDC (22), in the present study, amouse IgG2a version of the IgG1 EMab‑134 (14) was generated
by subcloning V H cDNA of EMab‑134 and CH mouse IgG2a
into pCAG‑Ble vector, and V L and CL cDNAs of EMab‑134
into pCAG‑Neo vector. The IgG2a version of EMab‑134 wasnamed 134‑mG2a. The sensitivity of 134‑mG2a in CHO/EGFR,
HSC‑2 and SAS cells was analyzed by flow cytometry. As
shown in Fig. 1, EMab‑134 and 134‑mG2a were equally effective at detecting CHO/EGFR, HSC‑2 and SAS cells using thismethod.
Subsequently, the sensitivities of EMab‑134 and 134‑mG2awere compared when used to probe lysates of CHO/EGFR,
HSC‑2 and SAS cells by western blot analysis. Notably,
134‑mG2a exhibited a relatively higher reactivity when
detecting their targets in cell lysates from CHO/EGFR and
HSC‑2 cells; both antibodies were faintly reactive against
targets in SAS cell lysates (Fig. 2). The molecular weight of
the EGFRs of HSC‑2 and SAS cells was smaller than that
expressed in CHO‑K1 cells, as PA‑EGFR‑RAP‑MAP, inwhich 3 peptide tags, such as PA tag, RAP tag, and MAP tag
were added, was transfected into the CHO‑K1 cells (16).
Immunohistochemical analysis revealed that both
EMab‑134 and 134‑mG2a detected membrane antigens in oral
cancer tissues (Fig. 3). 134‑mG2a exhibited higher staining
intensities when compared to the results from EMab‑134 inseveral oral cancer tissues with various levels of EGFR expression; intensities included 1+ (Fig. 3A), 2+ (Fig. 3B) and 3+(Fig. 3C). No staining was observed in tissues incubated withthe buffer control.
A kinetic analysis of the interactions of EMab‑134 and134‑mG2a with CHO/EGFR cells was then performed using
flow cytometry. As shown in Fig. 4, the dissociation constant(KD) for the interaction of EMab‑134 with CHO/EGFR cells
was 3.2x10 ‑9 M. By contrast, the K D for the interaction of
134‑mG2a with CHO/EGFR cells was 2.1x10‑9 M (Fig. 4). The
binding affinity of 134‑mG2a for CHO/EGFR cells was 1.5‑foldhigher than that of EMab‑134; taken together with the resultsfrom western blot analysis, this result suggests that the higher
binding affinity of 134‑mG2a may result in the higher sensitivity
observed in western blot and immunohistochemical analyses.
134‑mG 2a ‑mediated ADCC in oral cancer cell lines.
Subsequently, whether the newly‑developed 134‑mG2a wascapable of mediating ADCC against CHO/EGFR cells or oral
cancer cell lines, including HSC‑2 and SAS cells was examined.
As shown in Fig. 5A, 134‑mG2a elicited ADCC (66% cytotoxicity; P<0.01) against CHO/EGFR cells more effectively
than did control mouse IgG2a (23% cytotoxicity). By contrast,
EMab‑134 promoted no significant ADCC (23% cytotoxicity;n.s.) against CHO/EGFR cells compared to that observed in
response to control mouse IgG1 (23% cytotoxicity). Similarly,
134‑mG2a elicited ADCC (53% cytotoxicity; P<0.01) against
the HSC‑2 cells more effectively than did control mouse IgG2a(13% cytotoxicity) (Fig. 5B). By contrast, EMab‑134 elicited
no significant ADCC (14% cytotoxicity; n.s.) against the
HSC‑2 cells compared to that observed in response to mouse
IgG1 control (13% cytotoxicity). Furthermore, 134‑mG2a elicited higher ADCC (63% cytotoxicity; P<0.01) against the SAS
cells compared with that elicited by control mouse IgG2a (32%
cytotoxicity; Fig. 5C), while EMab‑134 elicited no significant

Figure 1. Flow cytometry with anti‑EGFR mAbs. EGFR was detected on
CHO/EGFR, HSC‑2, and SAS cells using EMab‑134, 134‑mG2a, or buffer
control, followed by secondary antibodies. Fluorescence data were collectedusing an SA3800 Cell Analyzer. EGFR, epidermal growth factor receptor;
mAbs, monoclonal antibodies.

ADCC (32% cytotoxicity; n.s.) against SAS cells compared tothat observed in response to control mouse IgG1 (33% cytotoxicity). Taken together, the novel mAb 134‑mG2a exhibited
significantly higher ADCC for all 3 EGFR‑expressing celllines featured in the present study; by contrast, no ADCC wasobserved in response to EMab‑134.
134‑mG 2a ‑mediated CDC in oral cancer cell lines. The
present study then examined whether 134‑mG2a induces CDC
in CHO/EGFR cells or in oral cancer cell lines, includingINTERNATIONAL JOURNAL OF MOlecular medicine 46: 1443-1452, 2020

1447

Figure 2. Western blot analysis using anti‑EGFR mAbs. Cell lysates of CHO‑K1, CHO/EGFR, HSC‑2 and SAS cells were subjected to gel electrophoresis andtransferred onto PVDF membranes. The membranes were probed with EMab‑134, 134‑mG2a, and anti‑β‑actin followed by peroxidase‑conjugated anti‑mouse
immunoglobulins. EGFR, epidermal growth factor receptor; mAbs, monoclonal antibodies.

Figure 3. Immunohistochemical analysis of oral cancers with anti‑EGFR mAbs. Formalin‑fixed paraffin‑embedded tissue sections from various oral cancers
were probed with EMab‑134, 134‑mG2a, or buffer control, followed by detection using the Envision+ kit; scale bar=100 µm. H&E, hematoxylin and eosin
staining. (A) 1+ staining pattern. (B) 2+ staining pattern. (C) 3+ staining pattern. EGFR, epidermal growth factor receptor; mAbs, monoclonal antibodies.

HSC‑2 and SAS cells. As shown in Fig. 6A, 134‑mG2a elicited a higher degree of CDC (46% cytotoxicity; P<0.01) in
CHO/EGFR cells compared with that elicited by control mouse
IgG2a (5.9% cytotoxicity). By contrast, EMab‑134 elicited nosignificant CDC (11% cytotoxicity; n.s.) against CHO/EGFR
cells compared to that observed in response to control mouse
IgG1 (7.4% cytotoxicity). Similarly, 134‑mG2a elicited a higher
degree of CDC (79% cytotoxicity; P<0.01) against HSC‑2
cells compared with that elicited by control mouse IgG2a(19% cytotoxicity; Fig. 6B). By contrast, EMab‑134 elicited
no significant CDC (20% cytotoxicity; n.s.) against the HSC‑2

cells compared to that observed in response to control mouse
IgG1 (19% cytotoxicity). Furthermore, 134‑mG2a eliciteda higher degree of CDC (60% cytotoxicity; P<0.01) against
SAS cells compared with that elicited by control mouse IgG2a(15% cytotoxicity; Fig. 6C). By contrast, EMab‑134 elicited
no significant CDC (28% cytotoxicity; n.s.) against the SAS
cells compared to that observed in response to control mouse
IgG1 (20% cytotoxicity). Taken together, these results demonstrated that 134‑mG2a promoted significantly higher levelsof CDC against all EGFR‑expressing cells evaluated in thisstudy; by contrast, EMab‑134 was not effective in this role.

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HOSONO et al: ANTITUMOR ACTIVITIES OF AN ANTI-EGFR mAb

Figure 4. Determination of the binding affinity of anti‑EGFR mAbs fortargets on CHO/EGFR cells using flow cytometry. CHO/EGFR cells weresuspended in 100 µl of serially diluted mAbs, followed by the additionof Alexa Fluor 488‑conjugated anti‑mouse IgG. Fluorescence data werecollected using an EC800 Cell Analyzer. EGFR, epidermal growth factor
receptor; mAbs, monoclonal antibodies.

As the ADCC/CDC activities of 134‑mG2a in oral cancer cells
were all potent and effective, this antibody may also exert
antitumor activity against oral cancer cells in vivo.
Antitumor activities of 134‑mG2a in the mouse xenograftsof CHO/EGFR cells. In the CHO/EGFR xenograft models,
134‑mG2a (100 µg), EMab‑134 (100 µg) and control mouse
IgG (100 µg) were injected intraperitoneally into the miceon days 1, 7 and 14 following the injection of CHO/EGFR
cells. The tumor volume was measured on days 7, 9, 14, 18
and 21 after the injection. The administration of 134‑mG2aresulted in a significant reduction in tumor development ondays 7 (P<0.01), 9 (P<0.01), 14 (P<0.05), 18 (P<0.01) and 21(P<0.01) compared to the mice treated with either EMab‑134
or control mouse IgG (Fig. 7). No significant differences intumor volume were observed in a comparison between the
EMab‑134‑ and control IgG‑treated mice on days 7, 9, 14,18, and 21. The administration of 134‑mG2a resulted in a 41%
reduction in tumor volume compared to the EMab‑134‑treatedmice on day 21 post‑injection. Furthermore, tumors from the134‑mG2a‑treated mice weighed significantly less than tumors
from the EMab‑134‑treated mice (50% reduction; P<0.01,
Fig. 8A). No significant differences in tumor weights wereobserved when comparing those from the EMab‑134‑ andcontrol mouse IgG‑treated mice (Fig. 8A). Tumors that were
resected from mice on day 21 are illustrated in Fig. 8B. Total
body weights did not differ significantly among the 3 groups(data not shown). Taken together, these results indicated that

Figure 5. Evaluation of ADCC elicited by anti‑EGFR mAbs. (A) ADCC
elicited by EMab‑134, 134‑mG2a, control IgG1, or control IgG2a targeting
CHO/EGFR cells. (B) ADCC elicited by EMab‑134, 134‑mG2a, control IgG1,
or control IgG2a targeting HSC‑2 cells. (C) ADCC elicited by EMab‑134,
134‑mG2a, control IgG1, or control mouse IgG2a targeting SAS cells. Values
shown are the means ± SEM. Asterisks indicate statistical significance(**P<0.01; n.s., not significant; one‑way ANOVA and Tukey‑Kramer's test).
ADCC, antibody‑dependent cellular cytotoxicity; EGFR, epidermal growth
factor receptor; mAbs, monoclonal antibodies.

the administration of 134‑mG2a effectively reduced the growthof CHO/EGFR xenografts.
Antitumor activities of 134‑mG2a in mouse xenografts of HSC‑2
oral cancer cells. In the HSC‑2 xenograft models, 134‑mG2a(100 µg), EMab‑134 (100 µg), or control mouse IgG (100 µg)
were injected intraperitoneally into mice on days 1, 7 and 14
after the HSC‑2 cell injections. Tumor volume was measured ondays 7, 9, 14 and 18. The administration of 134‑mG2a resulted insignificantly decreased tumor development on days 7 (P<0.01),
9 (P<0.01), 14 (P<0.01) and 18 (P<0.01) in comparison to the
EMab‑134‑treated mice (Fig. 9). No significant differenceswere observed between the EMab‑134‑ and control IgG‑treatedmice on days 7, 9, 14 and 18. The administration of 134‑mG2aINTERNATIONAL JOURNAL OF MOlecular medicine 46: 1443-1452, 2020

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Figure 7. Evaluation of antitumor activity of 134‑mG2a in CHO/EGFR
xenografts (tumor size). CHO/EGFR cells (5x106 cells) were injected subcutaneously into the left flank. After day 1, 100 µg of EMab‑134, 134‑mG2a, or
control mouse IgG in 100 µl PBS were injected intraperitoneally into mice;
additional antibodies were then injected on day 7 and day 14. Tumor volume
was measured on days 7, 9, 14, 18 and 21. Values shown are the means ± SEM.
Asterisks indicate statistical significance (**P<0.01, *P<0.05; n.s., not significant; one‑way ANOVA and Tukey‑Kramer's test). EGFR, epidermal growth
factor receptor; mAbs, monoclonal antibodies.

Figure 6. Evaluation of CDC elicited by anti‑EGFR mAbs. (A) CDC elicitedby EMab‑134, 134‑mG2a, control IgG1, or control IgG2a in target CHO/EGFR
cells. (B) CDC elicited by EMab‑134, 134‑mG2a, control IgG1, or control IgG2ain target HSC‑2 cells. (C) CDC elicited by EMab‑134, 134‑mG2a, control IgG1,
or control IgG2a in target SAS cells. Values shown are the means ± SEM.
Asterisks indicate statistical significance (**P<0.01; n.s. not significant;one‑way ANOVA and Tukey‑Kramer's test). CDC, complement‑dependent
cytotoxicity; EGFR, epidermal growth factor receptor; mAbs, monoclonal
antibodies.

resulted in a 57% reduction in tumor volume compared to the
EMab‑134‑treated mice on day 18 post‑injection. Tumors fromthe 134‑mG2a‑treated mice weighed significantly less than
the tumors from the EMab‑134‑treated mice (37% reduction;
P<0.01, Fig. 10A). No significant differences in tumor weight
were observed when comparing those from the EMab‑134‑ andcontrol mouse IgG‑treated mice. Tumors resected on day 18
are illustrated in Fig. 10B. Total body weights did not differ
significantly among the 3 groups (data not shown). These
results indicated that the administration of 134‑mG2a effectively
limited the growth of HSC‑2 cell xenografts.
Antitumor activities of 134‑mG2a in mouse xenografts of SASoral cancer cells. In the SAS xenograft models, 134‑mG2a(100 µg), EMab‑134 (100 µg), and control mouse IgG (100 µg)
were injected intraperitoneally into the mice on days 1, 7 andFigure 8. Evaluation of antitumor activity of 134‑mG2a in CHO/EGFR
xenografts (tumor weight). (A) Xenograft tumors were resected from eachmouse on day 21; tumor weights were determined. Values shown are themeans ± SEM. Asterisks indicate statistical significance (**P<0.01; n.s., notsignificant; one‑way ANOVA and Tukey‑Kramer's test). (B) Resected tumorsof CHO/EGFR xenografts from each group on day 21. Scale bar, 1 cm.
EGFR, epidermal growth factor receptor.

14 after SAS cell injections. Tumor volumes were measured ondays 7, 9, 14 and 18. The administration of 134‑mG2a resulted

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HOSONO et al: ANTITUMOR ACTIVITIES OF AN ANTI-EGFR mAb

Figure 9. Evaluation of antitumor activity of 134‑mG2a in HSC‑2 xenografts(tumor size). HSC‑2 (5x106 cells) were injected subcutaneously into the left
flanks of the mice. After day 1, 100 µg of EMab‑134, 134‑mG2a, or control
mouse IgG in 100 µl PBS were injected intraperitoneally into the mice.
Additional antibodies were then injected on days 7 and 14. Tumor volume
was measured on days 7, 9, 14 and 18. Values shown are the means ± SEM.
Asterisks indicate statistical significance (**P<0.01; n.s., not significant;one‑way ANOVA and Tukey‑Kramer's test).

Figure 11. Evaluation of antitumor activity of 134‑mG2a in SAS xenografts(tumor size). SAS (5x10 6 cells) were injected subcutaneously into the left
flanks of the mice. After day 1, 100 µg of EMab‑134, 134‑mG2a, or control
mouse IgG in 100 µl PBS were injected intraperitoneally into the mice.
Additional antibodies were injected on days 7 and 14. Tumor volume wasmeasured on days 7, 9, 14 and 18. Values shown are the means ± SEM.
Asterisks indicate statistical significance (**P<0.01; n.s., not significant;one‑way ANOVA and Tukey‑Kramer's test).

Figure 10. Evaluation of antitumor activity of 134‑mG2a in HSC‑2 xenografts(tumor weight). (A) Xenograft tumors were resected from each mouse on
day 18; tumor weights were determined. Values shown are the means ± SEM.
Asterisks indicate statistical significance (**P<0.01; n.s., not significant;one‑way ANOVA and Tukey‑Kramer's test). (B) Resected tumors of HSC‑2
xenografts from each group on day 18. Scale bar, 1 cm.

Figure 12. Evaluation of antitumor activity of 134‑mG2a in SAS xenografts(tumor weight). (A) Xenograft tumors were resected from each mouse on
day 18; tumor weights were determined. Values shown are the means ± SEM.
Asterisks indicate statistical significance (**P<0.01; n.s., not significant;one‑way ANOVA and Tukey‑Kramer's test). (B) Resected tumors of SAS
xenografts from each group on day 18. Scale bar, 1 cm.

in significantly reduced tumor development as determinedon days 7 (P<0.01), 9 (P<0.01), 14 (P<0.01) and 18 (P<0.01)

when compared to tumors from the EMab‑134‑treated mice
(Fig. 11). No significant differences between EMab‑134 andINTERNATIONAL JOURNAL OF MOlecular medicine 46: 1443-1452, 2020

control mouse IgG‑treated mice on days 7, 9, 14 and 18 wereobserved. The administration of 134‑mG2a resulted in a 70%
reduction in tumor volume on day 18 compared to the responses
observed among the EMab‑134‑treated mice. Tumors fromthe 134‑mG2a‑treated mice weighed significantly less than
the tumors from the EMab‑134‑treated mice (60% reduction;
P<0.01, Fig. 12A). No significant differences in tumor weight
were observed when comparing the tumors from the EMab‑134‑
and control mouse IgG‑treated mouse resected on day 18. The
tumors resected from the mice are shown in Fig. 12B. Total
body weights did not significantly differ among the three groups(data not shown). These results indicated that the administrationof 134‑mG2a effectively limited the growth of SAS xenografts.
Discussion
The authors of the present study previously established asensitive and specific anti‑EGFR mAb, EMab‑134 (mouse
IgG1), which is very useful for several applications, including
flow cytometry, western blot analysis and immunohistochemistry (14). This antibody could not be used to investigate
antitumor activity as the IgG1 subclass does not exhibit
ADCC/CDC activities. Therefore, EMab‑134 was converted
into 134‑mG2a (IgG2a subclass). It was demonstrated that134‑mG2a elicits both ADCC and CDC in vitro (Figs. 5 and 6),
and antitumor activities against both CHO/EGFR xenografts(Figs. 7 and 8) and OSCC xenografts (Figs. 9‑12) in vivo.
Importantly, the administration of 134‑mG2a efficiently
reduced the growth of OSCC xenografts at all time points
examined when compared to the responses to EMab‑134.
Nevertheless, only limited reductions in HSC‑2 and SAStumor volume were observed in response to the administration of 134‑mG2a, to 57 and 70%, respectively. These results
suggest that targeting EGFR with this antibody may not be
sufficient to eliminate most OSCCs. The authors have previously added EGF to HSC‑2 and SAS cell lines; however, thesecell lines did not respond to EGF stimulation and did not growwell compared to the control cells (21). The results indicatedthat 134‑mG2a and EMab‑134 antibodies could not neutralize
the EGF‑EGFR axis. Taken together, antitumor activities by
134‑mG2a were exerted by ADCC and CDC activities, notneutralization.
In a previous study, it was found that HER2 was expressedin oral cancers, and that the administration of an anti‑HER2
mAb (clone H 2Mab‑19, mouse IgG2b) resulted in antitumor
activity against HSC‑2 and SAS xenografts (23). By contrast,
Mirza et al (24) demonstrated that only one case out of
140 OSCCs was HER2‑positive; as such, the feasibility ofanti‑HER2 therapy for OSCC remains uncertain. In anotherstudy, the authors developed a sensitive and specific mAbagainst EGFR that recognized a distinct epitope (clone
EMab‑17, mouse IgG2a) and that elicited both ADCC and
CDC, as well as antitumor activity against HSC‑2 and SAS
xenografts (21). The extent of ADCC, CDC or antitumor
activities of EMab‑17 and 134‑mG2a were not yet compared,
nor was the binding epitope of EMab‑17 determined; further
investigations are warranted in order to select the optimalanti‑EGFR mAb for the treatment of OSCCs.
The authors previously converted an anti‑podocalyxin(PODXL) mAb of IgG1 subclass (PcMab‑47) into a1451

mouse IgG2a‑type mAb (47‑mG2a) to facilitate the evaluation of ADCC and CDC (25). The authors also developed
47‑mG2a‑f, a core fucose‑deficient variant of 47‑mG2a inorder to increase its ADCC. In vivo analysis revealed that
47‑mG2a‑f, but not 47‑mG2a, exerted antitumor activity in
HSC‑2 and SAS xenograft models at administered 3 times atdoses of 100 µg/mouse/week; these results indicated that acore fucose‑deficient anti‑PODXL mAb may also be usefulfor antibody‑based therapy against PODXL‑expressing
OSCCs. Moreover, a cancer‑specific mAb (CasMab) against
podoplanin (PDPN) was established, which is expressed ina number of types of cancer, including oral cancers (26). In
xenograft models of HSC‑2 cells, a mouse‑human chimericmAb, chLpMab‑23, exerted antitumor activity by engaging
human NK cells; these results suggest that chLpMab‑23 may be
advantageous for antibody therapy against PDPN‑expressing
oral cancers (27). Antibody regimens that focus on multiple
targets, including EGFR, HER2, PODXL and PDPN, may
ultimately be effective with the goal of conquering oral
cancers. In the future, cancer‑specific anti‑EGFR mAbs may
also be developed that can reduce the adverse effects of traditional antibody therapy.
Acknowledgements
The authors would like to thank Ms. Saori Handa, Ms. Saki
Okamoto and Mr. Yu Komatsu (Department of Antibody
Drug Development, Tohoku University Graduate School of
Medicine) for providing technical assistance with the in vitro
experiments, and Ms. Akiko Harakawa (Institute of Microbial
Chemistry (BIKAKEN), Numazu, Microbial Chemistry
Research Foundation) for providing technical assistance withthe animal experiments.
Funding
The present study was supported in part by the Japan Agency
for Medical Research and Development (AMED) under the
grant nos. JP20am0401013 (to YK), JP20am0101078 (to YK)
and JP20ae0101028 (to YK), and by the Japan Society for
the Promotion of Science (JSPS) Grants‑in‑Aid for Scientific
Research (KAKENHI) grant no. 17K07299 (to MKK),
grant no. 19K07705 (to YK), and grant no. 20K16322 (to MS).
Availability of data and materials
The datasets used and/or analyzed during the study areavailable from the corresponding author on reasonable request.
Authors' contributions
HHo, TO, JT, TN, MS TA, YS and MY performed the experiments. MKK analyzed the experimental data. MK, HHa andYK conceived and designed the present study and wrote themanuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Animal experiments for ADCC and the antitumor activity
were approved by the institutional committee for experiments

1452

HOSONO et al: ANTITUMOR ACTIVITIES OF AN ANTI-EGFR mAbof the Institute of Microbial Chemistry (Permit. no. 2019‑049
for ADCC assays, 2019‑046 for antitumor experiments). The
tissues used were from a purchased tissue microarray.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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