MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 4, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0022

Defucosylated Anti-Epidermal Growth Factor Receptor
Monoclonal Antibody 134-mG2a-f Exerts Antitumor
Activities in Mouse Xenograft Models of Dog Epidermal
Growth Factor Receptor-Overexpressed Cells

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Nami Tateyama,1,* Ren Nanamiya,1,* Tomokazu Ohishi,2,* Junko Takei,1 Takuro Nakamura,1Miyuki Yanaka,1 Hideki Hosono,1 Masaki Saito,3 Teizo Asano,1 Tomohiro Tanaka,1Masato Sano,1 Manabu Kawada,2 Mika K. Kaneko,1 and Yukinari Kato1,3,i

The epidermal growth factor receptor (EGFR) is a type I transmembrane protein, which is a member of thehuman epidermal growth factor receptor (HER) family of receptor tyrosine kinases. EGFR is a crucial mediatorof cell growth and differentiation and forms homodimers or heterodimers with other HER family members toactivate downstream signaling cascades. We previously established an anti-human EGFR (hEGFR) monoclonal
antibody (mAb), clone EMab-134 (mouse IgG1), by immunizing mice with the ectodomain of hEGFR. In thisstudy, the subclass of EMab-134 was converted from IgG1 to IgG2a (134-mG2a) and further defucosylated (134mG2a-f) to facilitate antibody-dependent cellular cytotoxicity (ADCC). Although 134-mG2a-f was developed
against hEGFR, it was shown to cross-react with dog EGFR (dEGFR) using flow cytometry. The dissociation
constant (KD) of 134-mG2a-f against dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells was determined byflow cytometry to be 3.3 · 10-9 M, indicating that 134-mG2a-f possesses a high binding affinity to dEGFR.
Analysis in vitro revealed that 134-mG2a-f contributed to high levels of ADCC and complement-dependent
cytotoxicity (CDC) in experiments targeting CHO/dEGFR cells. Furthermore, the in vivo administration of 134mG2a-f significantly inhibited the development of CHO/dEGFR in comparison with the results observed in
response to control mouse IgG. Taken together, the findings of this study demonstrate that 134-mG2a-f could be
useful as part of a therapeutic regimen for dEGFR-expressing canine cancers.
Keywords: EGFR, monoclonal antibody, ADCC, CDC, antitumor activity

IntroductionT

he epidermal growth factor receptor (EGFR) is atransmembrane glycoprotein that constitutes one of thefour members of the EGFR family of tyrosine kinase receptors.(1) The EGFR family consists of four different receptors
with a common structure: HER1 (EGFR/ErbB1), HER2
(ErbB2/neu), HER3 (ErbB3), and HER4 (ErbB4).(2,3) The
downstream signal pathway of EGFR includes Src kinase,signal transducers and activators of transcription, Ras/Raf/
mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which promote division, migration, antiapoptosis, oncogenesis, and/or cell cycle
progression.(4) EGFR is overexpressed and contributes to poor

prognosis in many cancers.(5–8) Therefore, several anti-EGFR
monoclonal antibodies (mAbs) have been developed and areused in clinical practice. Two anti-EGFR mAbs, cetuximab(Erbitux) and panitumumab (Vectibix), have been used to treat
unresectable advanced or recurrent colorectal cancer. Although there have been significant advances in treatment withmAbs for targets, including EGFR in humans like this, mAb
therapy for animal species has not been largely established.
In dogs, EGFR is overexpressed in multiple tumors aswell as humans.(9–14) Survival analysis showed that several
dog mammary tumors with EGFR overexpression decreased
disease-free and overall survival.(10) In vitro analysis exhibited that vandetanib (caprelsa), a tyrosine kinase inhibitor
targeting EGFR with antitumor effects, dose dependently

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Japan.
3
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
*These authors contributed equally to this work.
i
ORCID ID (https://orcid.org/0000-0001-5385-8201).
2

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178

inhibited EGFR phosphorylation and PI3K/Akt activation to
inhibit EGF-induced proliferation and invasion in dog
mammary carcinoma cell lines.(15) Thus, the development of
a novel anti-EGFR mAb for therapeutic and/or diagnostic use
for dog cancers is essential, but no mAb targeting dog EGFR(dEGFR) is available yet. By contrast, the sequences of human EGFR (hEGFR) and dEGFR are 91% identical; thus,
Singer et al. investigated whether cetuximab can also exert aneffect on dEGFR.(16) Consequently, cetuximab was shown toexert effects on cell viability and cell cycle in a dEGFRexpressing canine mammary carcinoma cell line, P114. For
the establishment of dog cancer treatment, we must investigate whether anti-hEGFR mAbs are useful against dEGFR.
We previously developed a novel anti-hEGFR mAb,
EMab-134,(17) by immunizing mice with the purified recombinant ectodomain of hEGFR (hEGFRec) from culture
supernatants of hEGFRec-overexpressed LN229 cells. ThismAb is useful for flow cytometry, Western blotting, andimmunohistochemical analyses to detect hEGFR. Furthermore, the IgG2a version of EMab-134, 134-mG2a, has antitumor
activities in mouse xenograft models of hEGFR-overexpressing
oral squamous cell carcinoma.(18) In this study, we defucosylated 134-mG2a (134-mG2a-f) to reinforce antibody-dependent
cellular cytotoxicity (ADCC). Then, we determined whether
134-mG2a-f exhibits ADCC and complement-dependent
cytotoxicity (CDC) in dEGFR-overexpressed cells and antitumor activities in mouse xenograft models of dEGFRoverexpressed cells.
Materials and Methods
Antibodies

Anti-hEGFR mAb EMab-134 (mouse IgG1, kappa) was
developed as previously described.(17) To generate 134-mG2a,
VH cDNA of EMab-134 and CH mouse IgG2a were subcloned
into the pCAG-Ble vector, and VL and CL cDNAs of EMab134 were subcloned into the pCAG-Neo vector (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan), respectively.(18) Vectors were transfected into BINDS-09 cells(FUT8-deficient ExpiCHO-S cells) using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc., Waltham,
MA).(19) The resulting mAb, 134-mG2a-f, was purified withProtein G-Sepharose (GE Health Care Bio-Sciences, Pittsburgh, PA). Mouse IgG (cat. no. I8765) and IgG2a (cat. no.
M7769) were purchased from Sigma-Aldrich (St. Louis, MO).

TATEYAMA ET AL.
Animals

All animal experiments were performed following relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. Animal experiments for
ADCC and antitumor activity were approved by the Institutional Committee for Experiments of the Institute ofMicrobial Chemistry (permit no. 2021–019 for ADCC assays, 2021–006 for antitumor experiments). Mice weremaintained in a specific pathogen-free environment(23C – 2C, 55% – 5% humidity) on an 11-hour light/13hour 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-week period of each experiment. The loss of original body weight toa point >25% and/or a maximum tumor size >3000 mm3
were identified as humane endpoints for euthanasia. Mice
were euthanized by cervical dislocation; death was verified
by respiratory and cardiac arrest.
Flow cytometry

CHO-K1 and CHO/dEGFR cells were harvested by brief
exposure to 0.25% trypsin/1 mM ethylenediamine tetraacetic
acid (EDTA; Nacalai Tesque, Inc.). After washing withblocking buffer: 0.1% bovine serum albumin in phosphatebuffered saline (PBS), cells were treated with 1 lg/mL of
134-mG2a-f or control blocking buffer for 30 minutes at 4Cfollowed by Alexa Fluor 488-conjugated anti-mouse IgG at adilution of 1:1000 (cat no. 4408S; Cell Signaling Technology,
Inc., Danvers, MA) for 30 minutes at 4C. Fluorescence data
were collected using the EC800 Cell Analyzer (Sony Corp.,
Tokyo, Japan).

Cell lines

The CHO-K1 cell line was obtained from the American
Type Culture Collection (Manassas, VA). dEGFRoverexpressed CHO-K1 cells (CHO/dEGFR) were establishedby the transfection of pCAG/3xRIEDL-dEGFR into CHO-K1
cells using the Neon transfection system (Thermo Fisher Scientific, Inc.).(20) The amino acid sequence of 3 · RIEDL
comprises 15 amino acids (RIEDLRIEDLRIEDL). CHO-K1
and CHO/dEGFR were cultured in Roswell Park Memorial
Institute (RPMI)-1640 medium (Nacalai Tesque, Inc., Kyoto,
Japan), supplemented with 10% heat-inactivated fetal bovine
serum (FBS; Thermo Fisher Scientific, Inc.), 100 U/mL of
penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.). Cells were cultured at 37C in
a humidified atmosphere containing 5% CO2.

FIG. 1. Production of 134-mG2a (mouse IgG2a) and 134mG2a-f (core-fucose-deficient mouse IgG2a) from EMab134 (mouse IgG1).

ANTITUMOR ACTIVITIES BY DEFUCOSYLATED ANTI-EGFR MAB
Determination of the binding affinity

CHO/dEGFR cells were suspended in 100 lL of seriallydiluted 134-mG2a-f (0.006–100 lg/mL) followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG (1:200;
Cell Signaling Technology, Inc.). Fluorescence data werecollected using the EC800 Cell Analyzer (Sony Corp.). The
dissociation constant (KD) was calculated by fitting binding
isotherms to built-in one-site binding models in GraphPad
Prism 8 (GraphPad Software, Inc., La Jolla, CA).
Antibody-dependent cellular cytotoxicity

aseptically and single-cell suspensions were obtained byforcing spleen tissues through a sterile cell strainer (352360;
BD Falcon, Corning, New York, NY) using a syringe. Erythrocytes were lysed with a 10 s exposure to ice-cold distilledwater. Splenocytes were washed with Dulbecco’s modified
Eagle’s medium (DMEM; Nacalai Tesque, Inc.) and resuspended in DMEM with 10% FBS; this preparation wasused as effector cells. Target tumor cells were labeled with
10 lg/mL Calcein AM (Thermo Fisher Scientific, Inc.) andresuspended in the same medium. The target cells (2 · 104
cells/well) were plated in 96-well plates and mixed with effector cells (effector/target cell ratio, 100), 100 lg/mL of
134-mG2a-f, or control mouse IgG2a. Following a 5-h incubation at 37C, the release of Calcein into the supernatant wasmeasured in each well. The fluorescence intensity was determined using a microplate reader (Power Scan HT; BioTek
Instruments, Inc., Winooski, VT) with an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

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A total of five female 5-week-old BALB/c nude mice
(weighing 14–17 g) were purchased from Charles River Laboratories, Inc., (Kanagawa, Japan). Spleen cells from fivemice were used as the source of NK cells for the evaluation of
ADCC, which has been reported previously.(18) Following
euthanasia by cervical dislocation, the spleens were removed

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FIG. 2. Flow cytometry using 134-mG2a-f. (A) CHO/dEGFR and CHO-K1 cells were treated with 134-mG2a-f or
buffer control, followed by secondary antibodies. Fluorescence data were collected using the EC800 Cell Analyzer.
(B) Determination of the binding affinity of 134-mG2a-f for CHO/dEGFR cells using flow cytometry. CHO/dEGFRcells were suspended in 100 mL of serially diluted 134-mG2a-f, followed by the addition of Alexa Fluor 488-conjugatedanti-mouse IgG. Fluorescence data were collected using the EC800 Cell Analyzer. EGFR, epidermal growth factor
receptor.

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Cytolytic activity (% lysis) was calculated using the equation% lysis = (E - S)/(M - S) · 100, where ‘‘E’’ is the fluorescence measured 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 ofEDTA.

TATEYAMA ET AL.

CHO/dEGFR was analyzed by flow cytometry. As shown in
Figure 2A, 134-mG2a-f detected CHO/dEGFR, but not parental CHO-K1 cells.
A kinetic analysis of the interactions of 134-mG2a-f with
CHO/dEGFR cells was performed by flow cytometry. As
shown in Figure 2B, the KD for the interaction of 134-mG2a-f
with CHO/dEGFR cells was 3.3 · 10-9 M, suggesting that
134-mG2a-f shows high affinity to CHO/dEGFR cells.

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Complement-dependent cytotoxicity

The cells (2 · 104 cells/well) were plated in 96-well plates
and mixed with rabbit complement (final dilution 1:10; LowTox-M Rabbit Complement; Cedarlane Laboratories, Hornby, Ontario, Canada) together with 100 lg/mL of 134-mG2a-f
or control mouse IgG2a. Following 5 hours of incubation at 37C, MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;
inner salt] assay was performed using a CellTiter 96 AQueous
assay kit (Promega, Madison, WI).

134-mG2a-f-mediated ADCC and CDC
in CHO/dEGFR cells

We investigated whether 134-mG2a-f was capable of mediating ADCC against CHO/dEGFR cell lines. As shown in
Figure 3A, 134-mG2a-f showed ADCC (31% cytotoxicity;p < 0.05) against CHO/dEGFR cells more effectively thancontrol mouse IgG2a (14% cytotoxicity). We then investigated whether 134-mG2a-f could mediate CDC against
CHO/dEGFR cell lines. As shown in Figure 3B, 134-mG2a-f

Antitumor activity of 134-mG2a in xenograftsof CHO/dEGFR cells

A total of 16 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/dEGFR cells (0.3 mL of 1.33 · 108
cells/mL in DMEM) were mixed with 0.5 mL BD Matrigel
Matrix Growth Factor Reduced (BD Biosciences, San Jose,CA); 100 lL of this suspension (5 · 106 cells) was injectedsubcutaneously into the left flanks of the mice. On day 6
postinoculation, 100 lg of 134-mG2a-f (n = 8), or control
mouse IgG (n = 8) in 100 mL PBS was injected intraperitoneally. Additional antibody inoculations were performed ondays 14 and 21. At 25 days following cell implantation,
all mice were euthanized by cervical dislocation; tumor
diameters and volumes were determined as previously
described.(18)
Statistical analyses

All data are expressed as mean – standard error of themean (SEM). Statistical analysis was conducted with
Welch’s t test for ADCC and CDC, ANOVA and Sidak’s
multiple comparisons tests for tumor volume and mouseweight, and Welch’s t test for tumor weight. All calculations
were performed using GraphPad Prism 7 (GraphPad Software, Inc.). A p-value of < 0.05 was considered statisticallysignificant.
Results
Production and characterization of 134-mG2a-f,
a mouse defucosylated IgG2a-type anti-EGFR antibody

A mouse IgG2a version of the IgG1 EMab-134(17) was
previously generated by subcloning VH cDNA of EMab-134
and CH mouse IgG2a into the pCAG-Ble vector, and VL and
CL cDNAs of EMab-134 into the pCAG-Neo vector (Fig. 1).
The IgG2a version of EMab-134 was named 134-mG2a.(18)The vectors of 134-mG2a were transfected into BINDS-09
cells,(19) and the resulting defucosylated 134-mG2a wasnamed 134-mG2a-f (Fig. 1). The sensitivity of 134-mG2a-f in

FIG. 3. Evaluation of ADCC and CDC elicited by 134mG2a-f. (A) ADCC elicited by 134-mG2a-f or control mouse
IgG2a targeting CHO/dEGFR cells. (B) CDC elicited by
134-mG2a-f or control mouse IgG2a targeting CHO/dEGFRcells. The values shown are the means – SEM. Asterisks
indicate statistical significance (*p < 0.05; Welch’s t test).
ADCC, antibody-dependent cellular cytotoxicity; CDC,
complement-dependent cytotoxicity.

ANTITUMOR ACTIVITIES BY DEFUCOSYLATED ANTI-EGFR MAB
Antitumor activities of 134-mG2a-f in the mouse
xenografts of CHO/dEGFR cells

In the CHO/dEGFR xenograft models, 134-mG2a-f(100 lg) and control mouse IgG (100 lg) were injected intraperitoneally into the mice on days 6, 14, and 21 following

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elicited a higher degree of CDC (56% cytotoxicity; p < 0.05)
in CHO/dEGFR cells compared with that elicited by control
mouse IgG2a (24% cytotoxicity). These results demonstrated
that 134-mG2a-f promoted significantly higher levels of
ADCC and CDC against dEGFR-overexpressed cells.

181

FIG. 4. Evaluation of antitumor activity of 134-mG2a-f in CHO/dEGFR xenografts. (A) CHO/dEGFR cells (5 · 106 cells)
were injected subcutaneously into the left flank. After day 6, 100 mg of 134-mG2a-f or control mouse IgG in 100 mL PBS was
injected intraperitoneally into mice; additional antibodies were then injected on days 14 and 21. The tumor volume wasmeasured on days 6, 11, 14, 18, 21, and 25 after the injection. Values shown are the mean – SEM. Asterisks indicate
statistical significance (**p < 0.01, *p < 0.05; n.s., not significant; ANOVA and Sidak’s multiple comparisons test).
(B) Tumors of CHO/dEGFR xenografts were resected from 134-mG2a-f and control mouse IgG groups. Tumor weight on
day 25 was measured from excised xenografts. Values are mean – SEM. Asterisk indicates statistical significance(**p < 0.01, Welch’s t-test). (C) Resected tumors of CHO/dEGFR xenografts from control mouse IgG and 134-mG2a-fgroups on day 25. Scale bar, 1 cm. (D) Body weights of the mice implanted with CHO/dEGFR xenografts were recorded ondays 6, 11, 14, 18, 21, and 25 (n.s.: not significant). (E) Body appearance of mice on day 25. Scale bar, 1 cm.

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the injection of CHO/dEGFR cells. The tumor volume wasmeasured on days 6, 11, 14, 18, 21, and 25 after the injection.
The administration of 134-mG2a-f resulted in a significant
reduction in tumor development on days 14 ( p < 0.05), 18( p < 0.01), 21 ( p < 0.01), and 25 ( p < 0.01) compared with thatof the control mouse IgG (Fig. 4A). The administration of 134mG2a-f resulted in a 90% reduction in tumor volume comparedwith that of the control mouse IgG on day 25 postinjection.
Furthermore, tumors from the 134-mG2a-f-treated miceweighed significantly less than those from the control mouse
IgG-treated mice (87% reduction; p < 0.01, Fig. 4B). Tumorsthat were resected from mice on day 25 are demonstrated in
Figure 4C. Total body weights did not differ significantly
among the two groups (Fig. 4D). The body appearances ofmice on day 25 are demonstrated in Figure 4E. Taken together,
these results indicated that the administration of 134-mG2a-f
effectively reduced the growth of CHO/dEGFR xenografts.
Discussion

In human disease treatment, various types of mAbs areavailable and great consequences have been achieved sofar.(21–23) By contrast, there is only one commerciallyavailable mAb, lokivetmab (Cytopoint) targeting interleukin31, which is used to treat atopic dermatitis in dogs.(24,25)
Lokivetmab treatment is effective in reducing itching and theseverity of skin disease in dogs with a good safety profile.(26)Thus, developing effective and useful mAbs for applying toveterinary medicine is desirable.
Gene amplification or protein overexpression of EGFR has
been detected in a wide range of human tumors and is significantly associated with tumor progression and poor prognosis.(27–32) The aberrant activation of EGFR initiates
multiple intracellular pro-oncogenic signaling pathways, including RAS/MAPK and Akt/PI3K, leading to cell proliferation, differentiation, and migration.(33) Consequently,
EGFR-targeted therapies are clinically applied as single
agents or in combination with chemotherapy for several human cancers.(34–37) Interestingly, EGFR is highly conserved
between humans and dogs, and it can enhance the malignant
property in several canine cancers.(15,38) These results
prompted us to develop a novel anti-EGFR mAb for therapeutic and/or diagnostic use for canine cancers.
The most important aim of this study was to demonstrate
the cytotoxic activity of 134-mG2a-f against dEGFRexpressing cells. Indeed, this novel anti-EGFR mAb caused
significant growth inhibition against CHO/dEGFR cells
in vivo (Fig. 4), suggesting that 134-mG2a-f is applicable for
antibody therapy against canine cancers. Since the cells examined in this study were only dEGFR-overexpressed cells,
further studies using various types of dEGFR-expressing
canine cancers are required to develop effective antibody
therapies for canine cancers.
Author Disclosure Statement

The authors have no conflict of interests.
Funding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under Grant
Numbers JP21am0401013 (to Y.K.) and JP21am0101078

TATEYAMA ET AL.

(to Y.K.), and by the Japan Society for the Promotion of
Science ( JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant nos. 21K15523 (to T.A.), 21K07168 (to M.K.K.),
20K16322 (to M. Sano), and 19K07705 (to Y.K.).
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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: May 11, 2021
Accepted: July 10, 2021