MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 2, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0040

Original Articles
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Antitumor Activities in Mouse Xenograft Modelsof Canine Mammary Gland Tumor by Defucosylated
Mouse-Dog Chimeric Anti-Epidermal Growth
Factor Receptor Antibody (E134Bf)
Guanjie Li,1 Hiroyuki Suzuki,1 Junko Takei,2,3 Teizo Asano,2 Masato Sano,2 Tomohiro Tanaka,2Hiroyuki Harada,3 Takuya Mizuno,4 Tomokazu Ohishi,5 Manabu Kawada,5Mika K. Kaneko,2 and Yukinari Kato1,2

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 (mAb), clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR(dEGFR). The defucosylated mouse IgG2a version of EMab-134 exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/
dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, we produced adefucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf), and the reactivity of E134Bf against a canine
mammary gland tumor cell line (SNP) was examined by flow cytometry. Furthermore, E134Bf highly exerted
ADCC and CDC for SNP cells. The administration of E134Bf with canine mononuclear cells significantly
suppressed the SNP xenograft growth. These results suggest that E134Bf 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, mouse-dog chimeric antibody, ADCC, CDC, antitumor activity

canine mammary tumors have been reported as complex
carcinomas that include both luminal and myoepithelial cells.
In contrast, ER-tumors are classified into simple carcinomas
arise from genomic aberrations.(3) Therefore, it is suggested
as a model for studying human breast cancer.
Epidermal growth factor receptor (EGFR) is a type I glycoprotein that plays crucial roles in promotion of cellproliferation and survival through activation of RAS-RAFMEK-ERK and PI3K/Akt pathways.(4) Overexpression andmutations of EGFR have been identified with various cancers
in both human and dog.(5–9) EGFR expression is associatedwith poor prognosis in canine mammary cancer.(10) Since

Introduction

C

anine mammary gland tumor is the most common
neoplasm in female dogs. It has several similarities withhuman breast cancer, including histological and molecularsubtypes, metastasis, and response to treatment.(1) Similar tohuman breast cancer, canine mammary gland tumors can beclassified into luminal A, luminal B, human epidermal
growth factor receptor 2 (HER2)-overexpressing, basal-like,or normal-like according to the immunohistochemistry results. Luminal A (estrogen receptor [ER]-positive and HER2negative) was the most frequent subtype.(2) ER-positive

Departments of 1Molecular Pharmacology and 2Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Japan.
3
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Bunkyo-ku, Japan.
4
Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
5Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Japan.

53

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human EGFR (hEGFR) and dog EGFR (dEGFR) have 91%
amino acid homology,(11) the therapeutic methods targeting
hEGFR is thought to be available for canine tumor treatment.
In our previous study, we developed an anti-hEGFR
monoclonal antibody (mAb), clone EMab-134 (mouse IgG1,
kappa).(12) EMab-134 can recognize both hEGFR anddEGFR.(13) The 134-mG2a, mouse IgG2a type of EMab-134,
exerted antitumor activities against human oral squamouscell carcinoma.(14) Furthermore, defucosylated type of 134mG2a (134-mG2a-f ) possesses antitumor effects against
dEGFR-overexpressing cells.(13)In this study, we investigated whether a defucosylatedmouse-dog chimeric anti-EGFR mAb (E134Bf) possesses
antibody-dependent cellular cytotoxicity (ADCC), complementdependent cytotoxicity (CDC), and antitumor activities using
a canine mammary gland tumor model.

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Materials and Methods
Cell lines

A canine mammary gland tumor cell line, SNP, wasobtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer at Tohoku University (Miyagi, Japan).(15) SNP was cultured in
RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan),
supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA),
100 units/mL of penicillin, 100 lg/mL streptomycin, and0.25 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 was developed as previously
described.(12) To generate E134B, we subcloned VH complementary DNA (cDNA) of EMab-134 and CH of dog IgGB
into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical
Corporation, Osaka, Japan), along with VL cDNA of EMab134 and CL cDNA of dog kappa light chain into the pCAGNeo vector (FUJIFILM Wako Pure Chemical Corporation),
respectively. The vector of E134B was transfected into
BINDS-09 cells (FUT8-deficient ExpiCHO-S cells) using the
ExpiCHO Expression System (Thermo Fisher Scientific,Inc.). The resulting mAb, E134Bf, was purified with Protein
G-Sepharose (GE Healthcare Biosciences, Pittsburgh, PA).
Dog IgG was purchased from Jackson ImmunoResearch, Inc.
(West Grove, PA).
Animals

All animal experiments were performed following relevant
guidelines and regulations to minimize animal suffering anddistress in the laboratory. Animal experiments for the antitumor activity were approved by the Institutional Committee
for Experiments of the Institute of Microbial Chemistry(permit No. 2021-021). Mice were maintained in a specificpathogen-free environment (23C – 2C, 55% – 5% humidity)
on an 11 hours light/13 hours dark cycle with food and watersupplied 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 theloss of original body weight to a point >25% and/or a max-

LI ET AL.

imum tumor size >3000 mm3 as humane endpoints for euthanasia. Mice were euthanized by cervical dislocation; death
was verified by respiratory and cardiac arrest.
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 in phosphate-buffered saline(PBS), cells were treated with 1 lg/mL of E134Bf, or control
blocking buffer for 30 minutes at 4C. Then, cells were incubated in FITC-conjugated anti-dog IgG at a dilution of
1:1000 (Thermo Fisher Scientific, Inc.) for 30 minutes at 4C.
Fluorescence data were collected using the EC800 Cell
Analyzer (Sony Corp., Tokyo, Japan).
Determination of binding affinity

SNP cells were suspended in 100 lL of serially diluted
E134Bf (0.006–100 lg/mL) followed by FITC-conjugatedanti-dog IgG (1:200). Fluorescence data were collected using
the EC800 Cell Analyzer. The dissociation constant (KD) was
calculated by fitting binding isotherms to built-in one-sitebinding models in GraphPad Prism 8 (GraphPad Software,Inc., La Jolla, CA).
ADCC

Canine mononuclear cells (MNC) were obtained from
Yamaguchi University and resuspended in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% FBS to be used
as effector cells. Target SNP 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 cells ratio, 50), 100 lg/mL of
E134Bf, or control dog IgG. Following 4.5-hour incubation at
37C, the release of Calcein 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 following thelysis 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

SNP cells were labeled with 10 lg/mL Calcein AM andresuspended in the medium. They were then plated in 96-wellplates at 2 · 104 cells/well with rabbit complement (final dilution 1:10; Low-Tox-M Rabbit Complement; Cedarlane
Laboratories, Hornby, Canada) and 100 lg/mL of E134Bf orcontrol dog IgG. 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.

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55

Antitumor activity of E134Bf in xenografts of SNP cells

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A total of 16 female BALB/c nude mice (5 weeks old,weighing 14–17 g) were purchased from Charles River Laboratories, Inc. (Kanagawa, Japan), 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 6 postinoculation, 100 lg of E134Bf (n = 8) orcontrol dog IgG (n = 8) in 100 mL PBS were injected intraperitoneally. Additional antibody inoculations were performedon days 15 and 23. Canine MNC (6.3 · 105 cells), which wereobtained from Yamaguchi University, were injected surrounding the tumors treated with both dog IgG and E134Bf on
days 6, 15, and 23. At 28 days following cell implantation, allmice were euthanized by cervical dislocation. Tumor diameters and volumes were determined as previously described.(13)
Statistical analyses

All data are expressed as mean – standard error of themean. Statistical analysis was conducted with Tukey’s test
for ADCC and CDC, and Welch’s t test for tumor weight.
Analysis of variance and Sidak’s multiple comparisons tests
were conducted for tumor volume and mouse weight. All
calculations were performed using GraphPad Prism 8(GraphPad Software, Inc.). A p-value of <0.05 was considered statistically significant.
Results
Flow cytometry analysis against a canine mammary
gland tumor cell line, SNP cells using E134Bf

In our previous study, an anti-hEGFR mAb, EMab-134,
recognized dEGFR-overexpressed CHO/dEGFR cells, indicating that EMab-134 crossreacts with dEGFR.(13) In thisstudy, we produced the defucosylated mouse-dog chimericmAb from EMab-134 (E134Bf) and demonstrated that
E134Bf detected SNP cells (Fig. 1A), indicating that E134Bf
detect endogenous dEGFR expressed on SNP cells.
Determination of the binding affinity

A kinetic analysis of the interactions of E134Bf with SNP
cells was performed via flow cytometry. As shown in
Figure 1B, the KD for the interaction of E134Bf with SNP
cells was 9.7 · 10-10 M, suggesting that E134Bf shows highaffinity for SNP cells.
E134Bf-mediated ADCC and CDC in SNP cells

We next investigated whether E134Bf has ADCC against
SNP cells using canine MNC as effector. As shown in
Figure 2A, E134Bf showed ADCC (37.0% cytotoxicity)
against SNP cells more potent than dog IgG (14.4% cytotoxicity) and PBS (10.2% cytotoxicity).
We then investigated whether E134Bf has CDC against
SNP cells. As shown in Figure 2B, E134Bf elicited a higher
degree of CDC (37.7% cytotoxicity) in SNP cells comparedwith that elicited by dog IgG (14.4% cytotoxicity) and PBS(9.4% cytotoxicity). These results demonstrated that E134Bf
exerts ADCC and CDC against dEGFR-expressing SNP cells.

FIG. 1. Flow cytometry using E134Bf. (A) SNP cells
were treated with E134Bf or buffer control, followed by
FITC-conjugated anti-dog IgG. (B) Determination of thebinding affinity of E134Bf for SNP cells using flow cytometry. SNP cells were suspended in 100 mL of seriallydiluted E134Bf, followed by the addition of FITCconjugated anti-dog IgG. Fluorescence data were collectedusing the EC800 Cell Analyzer.

Antitumor activities of E134Bf in the mouse
xenografts of SNP cells

In the SNP xenograft models, E134Bf (100 lg) and control
dog IgG (100 lg) were injected intraperitoneally into mice ondays 6, 15, and 23 following the injection of SNP cells. Canine MNC were also injected surrounding the tumors in bothgroups on days 6, 15, and 23. The tumor volume was measured on days 6, 12, 15, 17, 23, 26, and 28 after the injection.
The administration of E134Bf resulted in a significant reduction in tumor development on day 12 ( p < 0.05), day 15( p < 0.01), day 17 ( p < 0.01), day 23 ( p < 0.01), day 26( p < 0.01), and day 28 ( p < 0.01) compared with that of thecontrol dog IgG (Fig. 3).
The administration of E134Bf resulted in a 35% reductionof tumor volume compared with that of the control dog IgG
on day 28 postinjection. Furthermore, the tumor weight of theE134Bf-treated mice was significantly lower than that ofcontrol dog IgG-treated mice (28% reduction; p < 0.05,
Fig. 4A). The resected tumors are shown in Figure 4B. Total

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LI ET AL.

FIG. 3. Evaluation of tumor volume in SNP xenografts.
SNP cells (5 · 106 cells) were injected subcutaneously into
the left flank. On day 6, 100 mg of E134Bf or dog IgG in
100 mL PBS were injected intraperitoneally into mice; additional antibodies were then injected on days 15 and 23.
Canine mononuclear cells (6.3 · 105 cells), which were obtained from Yamaguchi University, were injected surrounding the tumors treated with both dog IgG and E134Bf
on days 6, 15, and 23. The tumor volume was measured ondays 6, 12, 15, 17, 23, 26, and 28 after the tumor inoculation. Values are means – SEM. Asterisks indicate statisticalsignificance (**p < 0.01, *p < 0.05, n.s.; ANOVA and Sidak’s
multiple comparisons test). ANOVA, analysis of variance.

FIG. 2. Evaluation of ADCC and CDC elicited by
E134Bf. (A) ADCC elicited by E134Bf, dog IgG, or PBS
targeting SNP cells. Asterisks indicate statistical significance (*p < 0.05, n.s.; Tukey’s test). (B) CDC elicited by
E134Bf, dog IgG, or PBS targeting SNP cells. Values aremeans – SEM. Asterisks indicate statistical significance(*p < 0.05, n.s.; 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.
body weights of the two groups did not change significantly(Fig. 5A). The mice on day 28 are shown in Figure 5B.
Taken together, these results indicate that the administration of E134Bf effectively suppresses the tumor growth of
SNP xenografts.
Discussion

This study demonstrated antitumor activities of a defucosylated mouse-dog chimeric mAb of anti-hEGFR mAb(E134Bf) on in vitro and in vivo in canine mammary glandtumor cells, which express endogenous dEGFR. EGFR is
one of the important oncoproteins that promotes tumor cellproliferation and survival.(4) Therapeutic agents targeting
EGFR, including kinase inhibitors and antibody drugs, havebeen developed and clinically applied to human.(16,17) The
anti-EGFR antibody drugs, including cetuximab and panitumumab, have similar binding affinities to EGFR, but have

FIG. 4. Evaluation of tumor weight in SNP xenografts.
(A) SNP xenografts were resected from E134Bf and dog
IgG groups. Tumor weight on day 28 was measured. Values
are means – SEM. Asterisk indicates statistical significance(*p < 0.05, Welch’s t test). (B) The appearance of resected
tumors from E134Bf and dog IgG groups. Scale bar, 1 cm.

DEFUCOSYLATED MOUSE-DOG CHIMERIC ANTI-EGFR MAB

57

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Approved drugs for human are often used for canine
cancer therapy due to their similarities.(29,30) EMab-134 wasoriginally developed using hEGFR as immunogen.(12) In caseof antibody drug, the development of chimeric antibody isessential to evaluate the effect in dog. In this study, we developed mouse-dog chimeric antibody and produced defucosylated form to potentiate the ADCC activity. E134Bf
possesses high affinity to dEGFR (Fig. 1B). Recently, we
have determined an epitope of EMab-134 between G378 toP386 of hEGFR(31) which is conserved in large part between
human and dog. Since E134Bf exerted ADCC and antitumor
effect in the presence of canine MNC (Figs. 2A and 3),
E134Bf is expected to apply the clinical use. Further studies
are required for the investigation of antitumor activity of
E134Bf to spontaneously develop canine cancer and the establishment of technique to diagnose canine cancer, including dEGFR expression.
Author Disclosure Statement

The authors have no conflict of interest.
Funding Information

FIG. 5. Evaluation of body weights in SNP xenografts.
(A) Body weights of mice implanted with SNP xenografts
were recorded on days 6, 12, 15, 17, 19, 23, 26, and 28 after
the tumor inoculation. (n.s.). (B) The appearance of micefrom E134Bf and dog IgG groups. Scale bar, 1 cm.
different epitopes on the EGFR.(18–20) These mAbs possess
higher affinity to EGFR than EGF and have neutralization
ability to inhibit the EGFR signaling pathways.(21)Basal type breast carcinoma includes triple negative breast
carcinoma (TNBC) characterized by the absence or low expression of ER, progesterone receptor, and HER2.(22) TNBC
has a poor prognosis compared to other breast cancer subtypes. Currently, molecular targeted drugs, including PD-L1/
PD-1 inhibitors, PARP inhibitors, and anti-TROP2 antibody
drug conjugates have received FDA approval.(23) Growing
number of evidences suggest that EGFR pathway plays
critical roles in the malignant progression of TNBC.(24,25)
EGFR activation/amplification is detected in *25%–50% of
TNBC.(26,27) EGFR inhibition by antibody drug and/or EGFR
tyrosine kinase inhibitors should be effective in the treatment
of EGFR-driven TNBC.
Although EGFR is overexpressed in TNBC, clinical trials
with EGFR inhibitors, including cetuximab, have been unsuccessful.(28) Novel approach to inhibit EGFR signaling isthought to be required. In this study, we could detect dEGFR
expression in SNP cells (Fig. 1A). Furthermore, E134Bf
exerts antitumor effects through ADCC and CDC activities(Figs. 2 and 3). Therefore, this model is useful to evaluate the
EGFR targeting drugs and/or combination therapy with antitumor drugs.

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under Grant
numbers: JP22ama121008 (to Y.K.), JP21am0401013 (toY.K.), and JP21am0101078 (to Y.K.), and by the Japan Society for the Promotion of Science ( JSPS) Grants-in-Aid for
Scientific Research (KAKENHI) grant numbers 21K20789(to T.T.), 21K15523 (to T.A.), 21K07168 (to M.K.K.),
20K16322 (to M.S.), 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
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: September 7, 2021
Accepted: February 14, 2022