MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 3, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0009

Short Communications
Open camera or QR reader andscan code to access this article
and other resources online.

Defucosylated Anti-Epidermal Growth Factor Receptor
Monoclonal Antibody Exerted Antitumor Activities in Mouse
Xenograft Models of Canine Mammary Gland Tumor

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Tomohiro Tanaka,1 Tomokazu Ohishi,2 Masaki Saito,3 Hiroyuki Suzuki,3Mika K. Kaneko,1 Manabu Kawada,2 and Yukinari Kato1,3

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification
and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal
antibody, clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR(dEGFR). The defucosylated mouse IgG2a version of EMab-134 (134-mG2a-f) exhibits antibody-dependent
cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed Chinese
hamster ovary-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In
this study, the reactivity of 134-mG2a-f against a canine mammary gland tumor cell line (SNP) was examinedby flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f highly exerted ADCC and CDC for
SNP. The administration of 134-mG2a-f significantly suppressed the SNP xenograft growth. These results
suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine mammary gland tumors,and could be valuable as part of an antibody treatment regimen for them.
Keywords: EGFR, monoclonal antibody, ADCC, CDC, canine breast cancer, antitumor activity

Introduction

F

emale breast cancer has the highest number ofworldwide new cases of all diagnosed cancers, with
2,261,419 cases (11.7% of new all cancer cases) and 684,996
deaths (6.9% of the total new cancer deaths) in 2020.(1)Breast cancer deaths have steadily decreased, but their diagnostic rates and deaths are globally high.(2)Canines are one of the animals that spontaneously develop
cancers, including breast cancers. In terms of histological,
genetic, and biological features, dog has strong similarities tohuman.(3–6) Dogs share carcinogenic factors, such as aging,
exposure to carcinogens, obesity, and environmental risk
factors with humans.(7,8) Dogs grow five to eight times fasterthan humans, making them suitable as models for age-related
gene profiling and pathogenesis research.(6)12
3The gene amplification of MYC oncogene and the loss oftumor suppressor gene PTEN have been found in both humanand canine breast cancers.(9) The tumor suppressor gene P53
abnormalities also have been found in several canine tumors,
including mammary tumors.(10,11) Dysregulations in thesegenes are often found in human cancers. Among mutations
found in human breast cancers, BRCA1, IGF2R, and USH2A
mutations have also been confirmed in dog cancer.(12) Estrogen receptor (ER)+ canine mammary tumors have beenreported as complex carcinomas that include both luminal
and myoepithelial cells. In contrast, ER-tumors are classified
into simple carcinomas arise from genomic aberrations.(12)Response to cancer treatment, acquisition of drug resistance, metastasis, and recurrence were similarly observed inboth dogs and humans.(13) Due to its similarity, human cancer
research is expected to be applied into canine.(7,14) It has been

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.
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.

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ANTITUMOR ACTIVITIES BY DEFUCOSYLATED ANTI-EGFR MAB

reported that inhibition of programmed cell death-1 (PD-1)
pathway is one of the therapeutic strategies in canine metastatic oral malignant melanoma.(15)
Epidermal growth factor receptor (EGFR) is a type I glycoprotein that forms a dimer by binding its ligand and regulates cell proliferation and survival. Overexpression andmutations of EGFR activate multiple intracellular cascades,such as RAS-MAPK and PI3K/AKT pathways.(16) EGFR isclosely associated with various cancer development andmalignancy in both human and dog.(17–21) EGFR expression
also correlates with poor prognosis in several canine cancers,
including mammary cancer.(22–24) Human EGFR (hEGFR)
and dog EGFR (dEGFR) have 91% amino acid homology.(25)
Therefore, the development of therapeutic methods targeting
EGFR is suitable as a cancer treatment for dog.
In our previous study, we developed an anti-hEGFR
monoclonal antibody (mAb), clone EMab-134 (mouse IgG1,
kappa).(26) The 134-mG2a, an IgG2a type of EMab-134, exerted antitumor activities in model of human oral squamouscell carcinoma.(27) Furthermore, a 134-mG2a-f, defucosylated type of 134-mG2a, possesses antitumor effects inmouse xenograft models of dEGFR-expressed cells.(28)The aim of this study is to investigate whether 134-mG2a-f
possesses antibody-dependent cellular cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC), and antitumor
activities using a canine mammary gland tumor model.
Materials and Methods
Cell lines

A canine mammary gland tumor cell line, SNP, was obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer at
Tohoku University (Miyagi, Japan).(29) SNP was cultured in
Roswell Park Memorial Institute-1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan), supplemented with 10% heatinactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA), 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.
Antibodies

Anti-hEGFR mAb EMab-134 (mouse IgG1, kappa) was
developed as previously described.(26) To generate recombinant EMab-134 (recEMab-134), we subcloned VH and CH
of cDNAs of EMab-134 into the pCAG-Neo vector, alongwith VL and CL cDNAs of EMab-134 into the pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan), respectively. To generate 134-mG2a, we subcloned
VH cDNA of EMab-134 and CH mouse IgG2a into the pCAGNeo vector, along with VL and CL cDNAs of EMab-134 into
the pCAG-Ble vector, respectively.(27) Vectors of EMab-134
and 134-mG2a were transfected into ExpiCHO-S cells and
BINDS-09 cells (FUT8-deficient ExpiCHO-S cells) using the
ExpiCHO Expression System (Thermo Fisher Scientific,Inc.), respectively.(28) The resulting mAbs, recEMab-134 and134-mG2a-f, were purified with Protein G-Sepharose(GE Health care Biosciences, Pittsburgh, PA).(28) Mouse IgG(cat. no. I8765) and IgG2a (cat. no. M7769) were purchasedfrom Sigma-Aldrich (St. Louis, MO).

143

Flow cytometry

SNP cells were harvested by brief exposure to 0.25%
trypsin/1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). After washing with blocking buffer of
0.1% bovine serum albumin (BSA) in phosphate-bufferedsaline (PBS), cells were treated with 1 lg/mL of recEMab134, 134-mG2a-f, or control blocking buffer for 30 minutes at
4C. Then, cells were incubated in Alexa Fluor 488conjugated anti-mouse IgG at a dilution of 1:1000 (cat no.
4408S; Cell Signaling Technology, Inc., Danvers, MA) for
30 minutes at 4C. Fluorescence data were collected using theEC800 Cell Analyzer (Sony Corp., Tokyo, Japan).
Determination of binding affinity

SNP cells were suspended in 100 lL of serially diluted
134-mG2a-f (0.006–100 lg/mL) followed by Alexa Fluor
488-conjugated anti-mouse IgG (1:200; Cell Signaling
Technology, Inc.). Fluorescence data were collected 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).
Immunocytochemical analysis

Subconfluent SNP cells cultured on acid-wash coverslips
were fixed in 4% paraformaldehyde in PBS for 10 minutes atroom temperature. After quenching with 50 mM NH4Cl in
PBSc/m (PBS containing 0.2 mM Ca2+ and 2 mM Mg2+) for 10
minutes, the cells were blocked with blocking buffer (PBSc/m
containing 0.5% BSA) for 30 minutes and incubated with
10 lg/mL of recEMab-134, 134-mG2a-f, or control blocking
buffer for 1 hour. Then, the cells were labeled with Alexa Fluor
488-conjugated anti-mouse IgG (1:400; Cell Signaling Technology, Inc.) for 45 minutes. The cell nuclei were stained with
4¢,6-diamidino-2-phenylindole (Thermo Fisher Scientific,Inc.). A fluorescence microscope BZ-X800 (Keyence, Osaka,
Japan) was used to acquire fluorescence images.
Animals

All animal experiments were performed following relevant
guidelines and regulations to minimize animal suffering anddistress in the laboratory. Animal experiments for ADCC andantitumor activity were approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (permit nos. 2021-028 for ADCC assays and 2021-021
for antitumor experiments). Mice were maintained in a specific pathogen-free environment (23C – 2C, 55% – 5% humidity) on an 11 hours light/13 hours dark cycle with foodand water supplied ad libitum across the experimental period.
Mice were monitored for health and weight every 2–5 days
during the 3-week period of each experiment. We determined
the loss of original body weight to a point >25% and/or amaximum tumor size >3000 mm3 as humane endpoints foreuthanasia. Mice were euthanized by cervical dislocation;
death was verified by respiratory and cardiac arrest.
Antibody-dependent cellular cytotoxicity

A total of five female 5-week-old BALB/c nude mice
(weighing 14–17 g) were purchased from Charles River

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Laboratories, Inc., (Kanagawa, Japan). Spleen cells from fivemice were used as the source of natural killer cells for theevaluation of ADCC, as previously reported.(28) Following euthanasia by cervical dislocation, the spleens were removed
aseptically, and a syringe was used to force spleen tissues
through a sterile cell strainer (352360; BD Falcon, Corning, New
York, NY) and obtain single-cell suspensions. Erythrocytes werelysed by 10-s exposure to ice-cold distilled water. Splenocytes
were washed with Dulbecco’s modified Eagle’s medium(DMEM; Nacalai Tesque, Inc.) and resuspended in DMEM with
10% FBS to be used as effector cells. SNP cells were labeledwith 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-wellplates and mixed with effector cells (effector/target cells ratio,50), 100 lg/mL of 134-mG2a-f or control mouse IgG2a. Following 4.5-hour incubation at 37C, the release of Calcein into
the supernatant was measured in each well. The fluorescenceintensity was determined using a microplate reader (Power
Scan HT; BioTek Instruments, Inc., Winooski, VT) with an
excitation wavelength of 485 nm and an emission wavelengthof 538 nm. Cytolytic activity (% lysis) was calculated as follows: % 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 of EDTA.

for ADCC and CDC, and Welch’s t test for tumor weight.
Analysis of variance (ANOVA) and Sidak’s multiple comparisons tests were conducted for tumor volume and mouseweight. All calculations were performed using GraphPad
Prism 8 (GraphPad Software, Inc.). A p-value of <0.05 wasconsidered statistically significant.

Complement-dependent cytotoxicity

Immunocytochemical analysis against canine
mammary gland tumor cell line, SNP cells
using recEMab-134 and 134-mG2a-f

SNP cells were labeled with 10 lg/mL Calcein AM (Thermo
Fisher Scientific, Inc.) and resuspended in the medium. They
were then plated in 96-well plates at 2 · 104 cells/well withrabbit complement (final dilution 1:10; Low-Tox-M Rabbit
Complement; Cedarlane Laboratories, Hornby, Ontario, Canada) and 100 lg/mL of 134-mG2a-f or control mouse IgG2a.
Following 4 hours of incubation at 37C, we measured Calcein
release into the supernatant for each well. Fluorescence intensity was calculated as described in the ADCC section above.
Antitumor activity of 134-mG2a-f in xenograftsof SNP 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. SNP 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 injected subcutaneously into the left flanks of the mice. On day 7 postinoculation, 100 lg of 134-mG2a-f (n = 8) or control mouse
IgG (n = 8) in 100 mL PBS were injected intraperitoneally.
Additional antibody inoculations were performed on days 11
and 18. At 25 days following cell implantation, all mice wereeuthanized by cervical dislocation. Tumor diameters andvolumes were determined as previously described.(28)
Statistical analyses

All data are expressed as mean – standard error of themean. Statistical analysis was conducted with Tukey’s test

Results
Flow cytometry analysis against canine mammary glandtumor cell line, SNP cells using recEMab-134
and 134-mG2a-f

In our previous study, an anti-hEGFR mAb, EMab-134,
recognized dEGFR-overexpressed CHO/dEGFR cells, indicating that EMab-134 crossreacts with dEGFR.(28) In thisstudy, recEMab-134 also detected canine mammary glandtumor cell line, SNP cells (Fig. 1A). Similarly, the defucosylated mouse IgG2a type of EMab-134, 134-mG2a-f, detected SNP cells (Fig. 1B), indicating that both recEMab-134
and 134-mG2a-f detect endogenous dEGFR-expressed on
SNP cells.
Determination of binding affinity

A kinetic analysis of the interactions of 134-mG2a-f with
SNP cells was performed via flow cytometry. As shown in
Figure 1C, the KD for the interaction of 134-mG2a-f with SNP
cells was 1.5 · 10-9 M, suggesting that 134-mG2a-f showshigh affinity for SNP cells.

RecEMab-134 and 134-mG2a-f were applied to immunocytochemistry in SNP cells. As shown in Figure 1D,
recEMab-134 and 134-mG2a-f, but not buffer control, visualized dEGFR in SNP cells, suggesting that recEMab-134
and 134-mG2a-f recognize endogenous dEGFR in SNP
cells.
134-mG2a-f-mediated ADCC and CDC in SNP cells

We next investigated whether 134-mG2a-f has ADCC
against SNP cells. As shown in Figure 2A, 134-mG2a-f
showed ADCC (46.8% cytotoxicity) against SNP cells morepotent than mouse IgG2a (12.9% cytotoxicity) and PBS (7.8%
cytotoxicity).
We then investigated whether 134-mG2a-f has CDC
against SNP cells. As shown in Figure 2B, 134-mG2a-f elicited a higher degree of CDC (59.5% cytotoxicity) in SNP
cells compared with that elicited by mouse IgG2a (27.3%
cytotoxicity) and PBS (18.5% cytotoxicity). These results
demonstrated that 134-mG2a-f exerts ADCC and CDC
against dEGFR-expressing SNP cells.
Antitumor activities of 134-mG2a-f in the mouse
xenografts of SNP cells

In the SNP xenograft models, 134-mG2a-f (100 lg) andcontrol mouse IgG (100 lg) were injected intraperitoneally
into mice on days 7, 11, and 18 following the injection of SNP
cells. The tumor volume was measured on days 7, 11, 14,
18, 21, and 25 after the injection. The administration of

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ANTITUMOR ACTIVITIES BY DEFUCOSYLATED ANTI-EGFR MAB

FIG. 1. Flow cytometry and immunocytochemistry using recEMab-134 and 134-mG2a-f. (A, B) SNP cells were treatedwith recEMab-134 (A) and 134-mG2a-f (B) or buffer control (A, B), followed by Alexa Fluor 488-conjugated anti-mouse
IgG. (C) Determination of the binding affinity of 134-mG2a-f for SNP cells using flow cytometry. SNP cells were suspended
in 100 mL of serially diluted 134-mG2a-f, followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG.
Fluorescence data were collected using the EC800 Cell Analyzer. (D) SNP cells were incubated with buffer control,
10 mg/mL of recEMab-134, or 10 mg/mL of 134-mG2a-f for 1 hour at room temperature. The cells were further incubatedwith Alexa Fluor 488-conjugated anti-mouse IgG and DAPI for 45 minutes at room temperature. Scale bars, 20 mm. DAPI,
4¢,6-diamidino-2-phenylindole; recEMab-134, recombinant EMab-134.

134-mG2a-f resulted in a significant reduction in tumor development on days 18 ( p < 0.05), 21 ( p < 0.05), and 25( p < 0.01) compared with that of the control mouse IgG(Fig. 3A). The administration of 134-mG2a-f resulted in a
31% reduction of tumor volume compared with that of thecontrol mouse IgG on day 25 postinjection. Furthermore, thetumor weight of the 134-mG2a-f-treated mice was significantly lower than that of IgG-treated mice (21% reduction;p < 0.05, Fig. 3B). Total body weights of the two groups did
not change significantly (Fig. 3C).

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TANAKA ET AL.

FIG. 2. Evaluation of ADCC and CDC elicited by 134mG2a-f. (A) ADCC elicited by 134-mG2a-f, mouse IgG2a, or
PBS targeting SNP cells. Values are means – SEM. Asterisks indicate statistical significance (*p < 0.05, **p < 0.01,
Tukey’s test). (B) CDC elicited by 134-mG2a-f, mouse IgG2a, or
PBS targeting SNP cells. Values are means – SEM. Asterisks
indicate statistical significance (*p < 0.05, Tukey’s test).
ADCC, antibody-dependent cellular cytotoxicity; CDC,
complement-dependent cytotoxicity; n.s., not significant; PBS,
phosphate-buffered saline; SEM, standard error of the mean.

Taken together, these results indicate that the administration of 134-mG2a-f effectively suppresses the tumor growthof SNP xenografts.
Discussion

EGFR is one of the important oncoproteins that promotes
tumor development.(16) Therapeutic agents targeting EGFR
have been developed and clinically applied.(30,31) The
EGFR-targeted antibody drugs, including cetuximab andpanitumumab,(32,33) possess similar binding affinities to
EGFR, but have different epitopes on the EGFR.(34–36)
These mAbs can inhibit the EGFR signaling pathways andinduce apoptosis in EGFR-expressing cancer cells and show
therapeutic potential for patients with advanced colorectal
cancer.(37,38)
Approved-drugs for human are often used to canine cancer
treatment due to their similarities.(7,39) Since higher expression levels of EGFR is involved in canine cancer malignancy
and poor prognosis, including lung, nasal, mammary, andtransitional cell carcinoma,(18,22–24) EGFR-targeting drugs
are considered as important therapeutic strategy. We havepreviously shown that the 134-mG2a-f has an antitumor effectagainst a dEGFR-overexpressed CHO model.(28) This study
also demonstrated antitumor activities of a defucosylated
IgG2a type of anti-hEGFR mAb (134-mG2a-f) on in vitro andin vivo in canine mammary gland tumor cells expressed endogenous dEGFR (Figs. 2 and 3). In the future study, we will

FIG. 3. Evaluation of antitumor activity of 134-mG2a-f in
SNP xenografts. (A) SNP cells (5 · 106 cells) were injectedsubcutaneously into the left flank. On day 7, 100 mg of 134mG2a-f or mouse IgG in 100 mL PBS were injected intraperitoneally into mice; additional antibodies were theninjected on days 11 and 18. The tumor volume was measured on days 7, 11, 14, 18, 21, and 25 after the injection.
Values are means – SEM. Asterisks indicate statistical significance (**p < 0.01, *p < 0.05, ANOVA and Sidak’s multiple comparisons test). (B) SNP xenografts were resectedfrom 134-mG2a-f and mouse IgG groups. Tumor weight on
day 25 was measured. Values are means – SEM. Asterisk
indicates statistical significance (*p < 0.05, Welch’s t test).
(C) Body weights of mice implanted with SNP xenografts
were recorded on days 7, 11, 14, 18, 21, and 25. ANOVA,
analysis of variance. n.s., not significant.

develop the antibody-based immunotherapeutic strategies
such as chimeric antigen receptor (CAR)-T cell therapy using
134-mG2a-f.(40)
Human epidermal growth factor receptor 2 (HER2) is oneof the oncogenes that belongs to the EGFR family of receptor
tyrosine kinases consisting of EGFR (HER1, ERBB1), HER2(ERBB2), HER3 (ERBB3), and HER4 (ERBB4).(41) HER2
is frequently confirmed to be overexpressed in human andcanine breast cancers.(42) Previously, we have also reported
that a specific mAb against HER2 (H2Mab-41) exerts strong

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ANTITUMOR ACTIVITIES BY DEFUCOSYLATED ANTI-EGFR MAB

antitumor activity against dog HER2-overexpressed cells.(43)Thus, the combination therapy of 134-mG2a-f with H2Mab41 might be effective for the treatment of canine cancer
clinically.
Combination therapy of cetuximab and pembrolizumab is
successful for recurrent or metastatic human head and necksquamous cell carcinomas.(44) Pembrolizumab is an immune
checkpoint inhibitor that recognizes immunosuppressive
molecule PD-1 expressed on T cells, and has been expanded
to various human cancers in recent years.(45) Not only immune checkpoint inhibitors but also CAR-T cell therapy isused to treat canine cancers.(46) Therefore, the combination ofanti-EGFR antibody therapy and immune checkpoint inhibitor is expected to be more effective in canine cancer treatment. Further studies are required for the investigation of
antitumor activity of 134-mG2a-f to spontaneously develop
canine cancer and the establishment of technique to diagnose
canine cancer, including dEGFR expression and immune
microenvironment.
Identified cell surface markers for the canine immune cellsubsets such as lymphoid, myeloid, and myeloid-derived
suppressor cells are approximately identical to humans.(47,48)
Therefore, dogs with an immune system similar to humans
have been focused as companion animals suitable for evaluation of cancer immunotherapies.(49) PD-1 is expressed on
total of 5%–10% CD4+ T cells and 20%–25% CD8+ T cells in
healthy dogs and is upregulated in canine cancer.(50) Sinceanti-PD-1 and anti-PD-L1 mAbs are used in canine cancer
treatment,(15,51,52) the combination of immune checkpoint
inhibitors with the other antibody drug is expected to be more
effective in canine cancer treatment in the future trials. Due toits genetical and histological similarity, canine cancer research can provide information feedback not only for development of canine cancer treatment but also for humancancer research.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under grantnos.: JP21am0401013 (to Y.K.) and JP21am0101078(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: February 6, 2022
Accepted: March 21, 2022