MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 2, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0059
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Antitumor Activities in Mouse Xenograft Models of Canine
Fibroblastic Tumor by Defucosylated Anti-Epidermal Growth
Factor Receptor Monoclonal Antibody

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Nohara Goto,1 Hiroyuki Suzuki,1 Tomokazu Ohishi,2 Akiko Harakawa,2 Guanjie Li,1 Masaki Saito,1 Junko Takei,3Tomohiro Tanaka,3 Teizo Asano,3 Masato Sano,3 Manabu Kawada,2 Mika K. Kaneko,3 and Yukinari Kato1,3

The epidermal growth factor receptor (EGFR) is involved in tumor malignancy through gene amplification
and/or protein overexpression. An anti-human EGFR (hEGFR) monoclonal antibody (clone EMab-134), which
explicitly detects hEGFR and dog EGFR (dEGFR), was previously developed. The defucosylated mouse IgG2a
version of EMab-134 (134-mG2a-f) exhibits antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, it was shown that 134-mG2a-f reacts
with a canine fibroblastic tumor cell line (A-72) using flow cytometry and immunocytochemistry. Furthermore,
134-mG2a-f exerted ADCC and CDC on A-72 cell line. The administration of 134-mG2a-f significantly inhibited the A-72 xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects on dEGFRexpressing canine fibroblastic tumors.
Keywords: EGFR, monoclonal antibody, ADCC, CDC, canine fibroblastic tumor, antitumor activity

Epidermal growth factor receptor (EGFR) is a type I
transmembrane protein that forms a dimer by binding its ligand
and regulating cell proliferation and survival. Overexpression
and mutations of EGFR activate multiple intracellular cascades.(10) EGFR is closely associated with different cancer
development and malignancy in humans and dogs.(11–15)
EGFR expression also correlates with poor prognosis in canine
mammary cancers.(16) Human EGFR (hEGFR) and dog EGFR(dEGFR) have 91% amino acid homology.(17) Therefore, thedevelopment of therapeutic methods targeting EGFR is suitable as a canine cancer treatment.
An anti-hEGFR monoclonal antibody (mAb), clone
EMab-134 (mouse IgG1, kappa), was previously developed.(18) The 134-mG2a, an IgG2a type of EMab-134, exerted
antitumor activities in a model of human oral squamous cell
carcinoma.(19) In addition, the 134-mG2a-f, defucosylated
type of 134-mG2a, possesses antitumor effects in mouse xenograft models of dEGFR-expressed cells.(20)IntroductionA

dog is an animal that spontaneously develops cancers, such as melanomas, breast cancers, and fibroblastic tumors.(1–4) In terms of genetic, histological, andbiological features, a dog has strong similarities to humans.
Dogs share carcinogenic factors, such as aging, obesity,
exposure to carcinogens, and environmental risk factors
with humans.(5,6) Dogs grow five to eight times faster thanhumans, making them suitable models for pathogenesis research and age-related gene profiling.(4) Response to cancer
treatment, the acquisition of drug resistance, and cancer
metastasis are similarly observed in dogs and humans.(7)Because of its similarity, human cancer research is expected
to be applied to dogs.(5,8) The inhibition of the programmed
cell death-1 (PD-1) pathway has been reported to be one of
the therapeutic strategies in canine metastatic oral malignant
melanoma.(9)123Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Shizuoka, Japan.
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.

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In this study, we investigated whether 134-mG2a-f possesses antibody-dependent cellular cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC), and antitumor
activities using a canine fibroblastic tumor xenograft model.
Materials and Methods

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Cell lines, antibodies, and animals

A canine fibroblast cell line (A-72) was obtained from theAmerican Type Culture Collection (Manassas, VA). A-72
was cultured in Dulbecco’s modified Eagle medium(DMEM; Nacalai Tesque, Inc., Kyoto, Japan), supplemented
using 10% heat-inactivated fetal bovine serum (FBS; Thermo
Fisher Scientific Inc., Waltham, MA), 100 U/mL of penicillin, 100 lg/mL of streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai Tesque, Inc.). Cells were cultured at 37C
in a humidified atmosphere containing 5% CO2.
An anti-hEGFR mAb, EMab-134, was developed as previously described.(18) To generate 134-mG2a, VH complementary DNA (cDNA) of EMab-134 and CH mouse IgG2a
was subcloned into the pCAG-Neo vector (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan), and VL and CL
cDNAs of EMab-134 were subcloned into the pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation).(19)The vectors of 134-mG2a were transfected into BINDS-09
cells (FUT8-deficient ExpiCHO-S cells) using the ExpiCHO
expression system (Thermo Fisher Scientific Inc.).(20) The
resulting mAbs (134-mG2a-f) were purified using Protein
G-Sepharose (GE Healthcare Biosciences, Pittsburgh,
PA).(20) Mouse IgG (cat. no. I8765) and mouse IgG2a (cat. no.
M7769) were bought from Sigma-Aldrich (St. Louis, MO).
Female BALB/c nude mice (5 weeks old, weighing 14–17 g)
were bought from Charles River Laboratories, Inc. The animal
experiments were conducted following relevant guidelines andregulations to minimize animal suffering and distress in thelaboratory. The institutional committee for experiments of theInstitute of Microbial Chemistry approved animal experiments
for ADCC and antitumor activity (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 11 hours
light/13 hours dark cycle with food and water supplied ad
libitum across the experimental period. Mice were monitored
for health and weight every 2–5 days during the 3 weeks
period of each experiment. The loss of original body weight
to a point >25% and/or a maximum tumor size >3000 mm3
were determined as humane endpoints for euthanasia. Mice
were euthanized by cervical dislocation; death was verified
through respiratory and cardiac arrest.
Flow cytometry

A-72 cells were harvested by brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA, Nacalai
Tesque, Inc.). After washing with 0.1% bovine serum albumin(BSA) blocking buffer in phosphate-buffered saline (PBS), cells
were treated with 1 lg/mL 134-mG2a-f or control blocking
buffer for 30 minutes at 4C. Then cells were incubated in Alexa
Fluor 488-conjugated 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 obtained using
the EC800 Cell Analyzer (Sony Corp., Tokyo, Japan).

GOTO ET AL.
Determination of binding affinity

A-72 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 obtained using theEC800 Cell Analyzer (Sony Corp.). The dissociation constant (KD) was calculated by fitting binding isotherms tobuilt-in one-site binding models in GraphPad Prism 8(GraphPad Software, Inc., La Jolla, CA).
Immunocytochemical analysis

Subconfluent A-72 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
PBS containing 0.2 mM Ca2+ and 2 mM Mg2+ (PBSc/m) for
10 minutes, the cells were blocked using a blocking buffer(PBSc/m containing 0.5% BSA) for 30 minutes and incubated with 10 lg/mL of 134-mG2a-f or control blocking
buffer for 1 hour. Then, the cells were labeled using Alexa
Fluor 488-conjugated anti-mouse IgG for 45 minutes. The
cell nuclei were stained using 4’,6-diamidino-2-phenylindole(DAPI; Thermo Fisher Scientific Inc.). A fluorescence microscope BZ-X800 (Keyence, Osaka, Japan) was used toobtain fluorescence images.
ADCC

ADCC assay was previously reported.(20–36) In brief,
spleen cells from five mice were used as the source ofmononuclear cells to evaluate ADCC. After euthanasia bycervical dislocation, the spleens were eliminated asepticallyand a syringe was used to force spleen tissue through a sterilecell strainer (352360; BD Falcon, Corning, New York, NY)
and obtain single-cell suspensions. Erythrocytes were lysedby 10 seconds exposure to ice-cold distilled water. Splenocytes were washed using DMEM and resuspended in DMEM
with 10% FBS to be used as effector cells.
A-72 cells were labeled with 10 lg/mL of Calcein AM(Thermo Fisher Scientific, Inc.) and resuspended in the same
medium. A-72 cells (2 · 104 cells/well) were plated in 96well plates and mixed with splenocytes (effector/target cells
ratio, 50), 100 lg/mL of 134-mG2a-f or control mouse IgG2a.
After 4.5 hours incubation at 37C, the amount of calcein
released into the supernatant was measured 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.
Cytolytic activity (% lysis) was calculated as follows: %lysis = (E - S)/(M - S) · 100, 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 after the lysisof all cells with a buffer containing 0.5% Triton X-100,
10 mM Tris-HCl (pH 7.4), and 10 mM EDTA.
CDC

CDC assay was previously reported.(20–36) In brief, A-72
cells were labeled with 10 lg/mL of Calcein AM (Thermo
Fisher Scientific, Inc.) and resuspended in the same medium.
A-72 cells were then plated in 96-well plates at 2 · 104

ANTITUMOR ACTIVITIES BY DEFUCOSYLATED ANTI-EGFR MAB

69

cells/well using rabbit complement (final dilution 1:10; LowTox-M Rabbit Complement; Cedarlane Laboratories, Hornby, Ontario, Canada) and 100 lg/mL of 134-mG2a-f orcontrol mouse IgG2a. After 4 hours of incubation at 37C, theamount of calcein released into the supernatant for each well
was measured.
Cytolytic activity (% lysis) was calculated as follows: %lysis = (E - S)/(M - S) · 100, 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 after the lysisof all cells with a buffer containing 0.5% Triton X-100,
10 mM Tris-HCl (pH 7.4), and 10 mM EDTA.

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Antitumor activity of 134-mG2a-f in xenograftsof A-72 cells

Sixteen female BALB/c nude mice were used in experiments once they reached 7 weeks of age. A-72 cells (0.3 mL
1.33 · 108 cells/mL in DMEM) were mixed with 0.5 mLof 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 ofthe mice.
On day 7 postinoculation, 100 lg of 134-mG2a-f (n = 8), or
control mouse IgG (n = 8) in 100 mL of PBS was injected
intraperitoneally. Additional antibody injections were administered on days 14 and 21. On day 25 after cell implantation, all mice were euthanized through cervical dislocation.
Tumor diameters and volumes were determined as previously
described.(20)
Statistical analyses

All data are expressed as mean – standard error of themean. Statistical analysis was conducted using Tukey’s test
for ADCC and CDC and Welch’s t-test for tumor weight.
Analysis of variance and Sidak’s multiple comparison tests
were performed for tumor volume and mouse weight. All
calculations were conducted using GraphPad Prism 8(GraphPad Software, Inc.). A p-value of <0.05 was considered statistically significant.
Results
Flow cytometry analysis against canine fibroblastic
tumor cell line, A-72 cells using 134-mG2a-f

In our previous study, an anti-hEGFR mAb, EMab-134,
recognized CHO/dEGFR cells, showing that EMab-134
cross-reacts with dEGFR.(20) In this study, the defucosylatedmouse IgG2a type of EMab-134 (134-mG2a-f) detected A-72
cells (Fig. 1A), showing that 134-mG2a-f detects endogenousdEGFR expressed on A-72 cells.

FIG. 1. Flow cytometry and immunocytochemistry using
134-mG2a-f. (A) Flow cytometry using A-72 cells and134-mG2a-f. (B) Determination of the binding affinity of 134mG2a-f for A-72 cells using flow cytometry. (C) Immunocytochemical analyses using A-72 cells and 134-mG2a-f.
DAPI, 4’,6-diamidino-2-phenylindole; dEGFR, dog epidermal growth factor receptor.

Determination of binding affinity

Immunocytochemical analysis against A-72 cells
using 134-mG2a-f

A kinetic analysis of the interactions of 134-mG2a-f with
A-72 cells was conducted using flow cytometry. As shown in
Figure 1B, the KD for the interaction of 134-mG2a-f with
A-72 cells was 1.1 · 10-9 M, suggesting that 134-mG2a-f
shows a high affinity for A-72 cells.

The 134-mG2a-f was applied to immunocytochemistry in
A-72 cells. As shown in Figure 1C, 134-mG2a-f, but notbuffer control, visualized dEGFR in A-72 cells, suggestingthat 134-mG2a-f recognizes endogenous dEGFR in A-72
cells.

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GOTO ET AL.

134-mG2a-f-mediated ADCC and CDC in A-72 cells

Next, it was investigated whether 134-mG2a-f has ADCC
against A-72 cells. As indicated in Figure 2A, 134-mG2a-f
showed ADCC (15.8% cytotoxicity) against A-72 cells,
which is more potent than mouse IgG2a (5.6% cytotoxicity)and PBS (4.0% cytotoxicity).
It was then investigated whether 134-mG2a-f has CDC
against A-72 cells. As shown in Figure 2B, 134-mG2a-f elicited a higher degree of CDC (43.2% cytotoxicity) in A-72
cells than that elicited by mouse IgG2a (36.1% cytotoxicity)and PBS (31.8% cytotoxicity). These results showed that
134-mG2a-f exerts ADCC and CDC against dEGFRexpressing A-72 cells.

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Antitumor activities of 134-mG2a-f in the mouse
xenografts of A-72 cells

In the A-72 xenograft models, 134-mG2a-f (100 lg) andcontrol mouse IgG (100 lg) were injected intraperitoneally intomice on days 7, 14, and 21 after the injection of A-72 cells. The
tumor volume was measured on days 7, 11, 14, 18, 21, and 25
postinoculation. The administration of 134-mG2a-f caused asignificant reduction in tumor development on days 11( p < 0.01), 14 ( p < 0.01), 18 ( p < 0.01), 21 ( p < 0.01), and 25( p < 0.01) compared with that of the control mouse IgG(Fig. 3A).

FIG. 3. Evaluation of antitumor activity of 134-mG2a-f in
A-72 xenografts. (A) A-72 cells were subcutaneously injected into the left flank. On day 7, 100 mg 134-mG2a-f ormouse IgG in 100 mL PBS was injected intraperitoneally
into mice; additional antibodies were then injected on days
14 and 21. 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,
n.s.; ANOVA and Sidak’s multiple comparisons test).
(B) A-72 xenografts were resected from 134-mG2a-f andmouse IgG groups. Tumor weight on day 25 was measured.
Values are means – SEM. Asterisk indicates statisticalsignificance (**p < 0.01, Welch’s t-test). (C) Body weightsof mice implanted with A-72 xenografts were recorded ondays 7, 11, 14, 18, 21, and 25 (n.s.). ANOVA, analysis ofvariance; n.s., not significant.
FIG. 2. Evaluation of ADCC and CDC elicited by 134mG2a-f. (A) ADCC elicited by 134-mG2a-f, mouse IgG2a, or
PBS targeting A-72 cells. Asterisks indicate statistical significance (*p < 0.05, n.s.; Tukey’s test). (B) CDC elicited by
134-mG2a-f, mouse IgG2a, or PBS targeting A-72 cells. Values are means – SEM. Asterisks indicate statistical significance (**p < 0.01, *p < 0.05, n.s.; Tukey’s test). ADCC,
antibody-dependent cellular cytotoxicity; CDC, complementdependent cytotoxicity; n.s., not significant; PBS, phosphatebuffered saline; SEM, standard error of the mean.

The administration of 134-mG2a-f caused 52% reduction
in tumor volume compared with that of the control mouse
IgG on day 25 postinjection. Furthermore, the tumor weightof the 134-mG2a-f-treated mice was significantly lower thanthat of IgG-treated mice (48% reduction; p < 0.01, Fig. 3B).
The total body weights of the two groups did not significantly
change (Fig. 3C). Altogether, these results indicate that theadministration of 134-mG2a-f effectively suppresses tumor
growth of A-72 xenografts.

ANTITUMOR ACTIVITIES BY DEFUCOSYLATED ANTI-EGFR MAB

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Discussion

EGFR is an essential oncoprotein, which promotes tumor
development.(10) Therefore, therapeutic agents targeting
EGFR have been developed.(37,38) The EGFR-targeted antibody drugs, including panitumumab and cetuximab,(39,40)
have similar binding affinities to EGFR but different epitopes
on the EGFR.(41–43) These mAbs can inhibit the EGFR signaling pathways and induce apoptosis in EGFR-expressing
cancer cells and exhibit therapeutic potential for patients withadvanced colorectal cancer.(44,45)
Approved drugs for humans are often used for canine
cancer treatment due to their similarities.(5,46) Because
EGFR is involved in canine cancer malignancy and poor
prognosis, EGFR-targeting drugs are considered an important therapeutic strategy. It has been previously shown thatthe 134-mG2a-f has an antitumor effect against a dEGFRoverexpressed CHO-K1 model.(20) This study also demonstrated antitumor activities of a defucosylated IgG2a type ofanti-hEGFR mAb (134-mG2a-f) in vitro and in vivo on canine fibroblastic tumor cells expressing endogenous dEGFR(Figs. 2 and 3).
The combination therapy of cetuximab and pembrolizumab is successful for human head and neck squamous cell
carcinomas.(47) Pembrolizumab is an immune checkpoint
inhibitor that recognizes immunosuppressive molecule PD-1
expressed on T cells and has been expanded to various human cancers recently.(48) Immune checkpoint inhibitorsand chimeric antigen receptor-T cell therapy are also used totreat canine cancers.(49) Therefore, the combination of antiEGFR mAb therapy and immune checkpoint inhibitors is
expected to be more effective in canine cancer treatment.
Further studies are needed to investigate the antitumor
activity of 134-mG2a-f to spontaneously develop canine
cancers.
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 Grant
Numbers: 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, Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: November 25, 2021
Accepted: January 25, 2022