MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 1, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0036

Original Articles
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Defucosylated Anti-Epidermal Growth Factor Receptor
Monoclonal Antibody (134-mG2a-f) Exerts Antitumor
Activities in Mouse Xenograft Modelsof Canine Osteosarcoma
Ren Nanamiya,1 Junko Takei,1 Tomokazu Ohishi,2 Teizo Asano,1 Tomohiro Tanaka,1 Masato Sano,1Takuro Nakamura,1 Miyuki Yanaka,1 Saori Handa,1 Nami Tateyama,1 Yasuhiro Harigae,3 Masaki Saito,3Hiroyuki Suzuki,3 Manabu Kawada,2 Mika K. Kaneko,1 and Yukinari Kato1,3

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein. Although EGFR is physiologically essential in normal cells, it contributes to tumor malignancy through gene amplification and/or protein
overexpression, which augment signaling cascades in tumor cells. We previously developed an anti-human
EGFR (hEGFR) monoclonal antibody (mAb), EMab-134 (mouse IgG1, kappa), which detects hEGFR and dog
EGFR (dEGFR) with high sensitivity and specificity. The mouse IgG2a version of EMab-134 (134-mG2a) has
antitumor effects toward mouse xenografts of hEGFR-expressing oral squamous cell carcinomas. Furthermore,
134-mG2a-f, the defucosylated version of 134-mG2a, exhibits antibody-dependent cellular cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells andantitumor activities in mouse xenografts of CHO/dEGFR cells. Herein, the reactivity of 134-mG2a-f against
canine cancer cells with endogenous dEGFR was first examined by flow cytometry and immunocytochemistry.
In vitro analysis demonstrated that 134-mG2a-f highly exerted ADCC and CDC for a canine osteosarcoma cellline, D-17, which expresses endogenous dEGFR. Moreover, in vivo administration of 134-mG2a-f significantly
suppressed the development of D-17 compared with the results in response to control mouse IgG. These results
suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine cancers, and could bevaluable as part of an antibody treatment regimen for them.
Keywords: EGFR, monoclonal antibody, ADCC, CDC, canine osteosarcoma, antitumor activity

gration, and inhibit apoptosis. EGFR gene mutation is mostcommon in lung cancer,(4) and overexpression is observed inmany tumors, such as osteosarcoma (OS),(5,6) glioblastoma,(7) breast cancer,(8) lung cancer,(4) and colorectal cancer,(9) which contributes to tumor malignancy by augmenting
the above signaling pathways.
OS is the most common primary bone tumor in dogs,
leading to metastasis.(10) The incidence of OS in dogs is 27
times higher than that in humans, with a 1-year survival rateof only about 45%.(11) Although therapy options for canine

IntroductionT

he epidermal growth factor receptor (EGFR) is a
type-I transmembrane glycoprotein and a receptor tyrosine kinase.(1,2) EGFR can form homodimers or heterodimers with other EGFR family members, including HER2
(ErbB2/neu), HER3 (ErbB3), and HER4 (ErbB4). These dimers activate various signaling pathways, including Ras/
Raf/MEK/ERK, PI3K-AKT-mTOR, and JAK-STAT pathways.(3) These pathways promote cell proliferation and mi-

1
2
3

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Microbial Chemistry Research Foundation, Institute of Microbial Chemistry (BIKAKEN), Numazu-shi, Japan.
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.

1

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2

OS include surgery (limb amputation or limb-sparing surgery), radiotherapy, and chemotherapy,(12,13) they are notsufficiently effective. Therefore, the development of therapeutic strategies for canine OS is still necessary.
Previously, we developed a novel anti-human EGFR(hEGFR) monoclonal antibody (mAb), EMab-134 (mouse
IgG1, kappa), by immunizing mice with the purified recombinant ectodomain of hEGFR (hEGFRec) from culture supernatants of hEGFRec-overexpressed human glioblastoma
LN229 cells.(14) EMab-134 can be used in flow cytometry,
Western blotting, and immunohistochemical analyses. The
mouse IgG2a version of EMab-134 (134-mG2a) exerts antitumor activities in mouse xenograft models of hEGFRexpressing oral squamous cell carcinoma.(15) In addition, we
produced the defucosylated version of 134-mG2a, 134-mG2a-f,
to augment antibody-dependent cellular cytotoxicity(ADCC).(16) 134-mG2a-f exhibits ADCC and complementdependent cytotoxicity (CDC) in dog EGFR (dEGFR)overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor
activities in mouse xenograft models of CHO/dEGFRcells.(16) In the present study, we investigated antitumor activities of 134-mG2a-f against the D-17 canine tumor cell line
endogenously expressed dEGFR.
Materials and Methods
Antibodies

Anti-hEGFR mAb EMab-134 (mouse IgG1, kappa) was
developed as previously described.(14) 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.(15) 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., Waltham, MA), respectively.(16)The resulting mAbs, recEMab-134 and 134-mG2a-f, were
purified with Protein G-Sepharose (GE Healthcare Biosciences, Pittsburgh, PA).(16) Mouse IgG (Cat. No. I8765) and
IgG2a (Cat. No. M7769) were purchased from Sigma-Aldrich(St. Louis, MO).
Cell lines

A canine OS cell line, D-17, was obtained from the American
Type Culture Collection (Manassas, VA). D-17 was cultured in
MEM (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with
10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher
Scientific, Inc.), 1 mM of sodium pyruvate, 100 units/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.
Animals

All animal experiments were performed following relevant
guidelines and regulations to minimize animal suffering anddistress in the laboratory. Animal experiments for ADCC and

NANAMIYA ET AL.

antitumor 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-hour light/13-hour dark cycle with food andwater 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.
Flow cytometry

D-17 cells were harvested by brief exposure to 0.25%
trypsin/1 mM ethylenediaminetetraaceticacid (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).
Immunocytochemical analysis

D-17 cells were fixed in 4% paraformaldehyde in PBS for
10 minutes, quenched with 50 mM NH4Cl in PBSc/m (PBS
supplemented with 0.2 mM Ca2+ and 2 mM Mg2+) for 10
minutes, and treated with blocking buffer (PBSc/m supplemented with 0.5% BSA) for 30 minutes. Then, the cells wereincubated with 10 lg/mL of recEMab-134 or 134-mG2a-f orcontrol blocking buffer for 1 hour followed by Alexa Fluor
488-conjugated anti-mouse IgG (1:400) and DAPI (Thermo
Fisher Scientific, Inc.) for 45 minutes. Fluorescence images
were acquired with a 40 · objective on a fluorescence microscope BZ-X800 (Keyence, Osaka, Japan).
Determination of binding affinity

D-17 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). Fluorescence data
were collected using the EC800 Cell Analyzer. 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

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, as previously reported.(16) Following euthanasia bycervical dislocation, the spleens were removed aseptically,
and a syringe was used to force spleen tissues through asterile cell strainer (352360; BD Falcon, Corning, New York,NY) and obtain single-cell suspensions. Erythrocytes werelysed by a 10-second exposure to ice-cold distilled water.

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ANTITUMOR ACTIVITIES BY DEFUCOSYLATED ANTI-EGFR MAB

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. Target
tumor cells were labeled with 10 lg/mL Calcein AM (Thermo
Fisher Scientific, Inc.) and resuspended in the same medium.
The target cells (2 · 104 cells/well) were plated in 96-wellplates and mixed with effector cells (effector/target cell ratio,50), 100 lg/mL of 134-mG2a-f, or control mouse IgG2a.
Following a 4.5-hour incubation at 37C, the release ofCalcein 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 fluorescence measured in combined cultures oftarget and effector cells, ‘‘S’’ is the spontaneous fluorescenceof target cells only, and ‘‘M’’ is the maximum fluorescencemeasured following the lysis of all cells with a buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4), and10 mM of EDTA.

3

Results
Flow cytometry analysis against canine OS D-17 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.(16) In thisstudy, recEMab-134 also detected canine OS D-17 cells(Fig. 1A). Similarly, the defucosylated mouse IgG2a type of
EMab-134 (134-mG2a-f) detected D-17 cells (Fig. 1B), indicating that both recEMab-134 and 134-mG2a-f could detect
the endogenous dEGFR expressed on D-17 cells.
Immunocytochemical analysis against canine OS D-17
cells using recEMab-134 and 134-mG2a-f

We also investigated whether recEMab-134 and 134mG2a-f were available for immunocytochemistry in D-17
cells. We found that recEMab-134 and 134-mG2a-f, but notbuffer control, labeled dEGFR in D-17 cells (Fig. 1C). The
result suggests that both recEMab-134 and 134-mG2a-f selectively recognize endogenous dEGFR in D-17 cells.

Complement-dependent cytotoxicity

Determination of binding affinity

Target tumor cells were labeled with 10 lg/mL of Calcein
AM and resuspended in the medium. They were then plated
in 96-well plates at 2 · 104 cells/well with rabbit 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 wascalculated as described in the Antibody-Dependent Cellular
Cytotoxicity section above.

A kinetic analysis of the interactions of 134-mG2a-f with
D-17 cells was performed via flow cytometry. As shown in
Figure 1D, the KD for the interaction of 134-mG2a-f with
D-17 cells was 9.0 · 10-10 M, suggesting that 134-mG2a-f
shows high affinity for D-17 cells.

Antitumor activity of 134-mG2a-f in xenograftsof D-17 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. D-17 cells (0.3 mL of 1.33 · 108 cells/mL in
DMEM) were mixed with 0.5 mL of 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) orcontrol mouse IgG (n = 8) in 100 mL of PBS was injected
intraperitoneally. Additional antibody inoculations were
performed on days 11 and 18. At 25 days following cell
implantation, all mice were euthanized by cervical dislocation. Tumor diameters and volumes were determined aspreviously described.(16)
Statistical analysis

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.
ANOVA and Sidak’s multiple comparisons tests were conducted for tumor volume and mouse weight. All calculations
were performed using GraphPad Prism 8. A p-value of <0.05
was considered statistically significant.

134-mG2a-f-mediated ADCC and CDC in D-17 cells

We investigated whether 134-mG2a-f was capable of mediating ADCC against D-17 cells. As shown in Figure 2A,
134-mG2a-f showed ADCC (14.4% cytotoxicity; p < 0.05)
against D-17 cells more effectively than did the control
mouse IgG2a (3.8% cytotoxicity) and the control PBS (2.1%
cytotoxicity).
We then investigated whether 134-mG2a-f could mediate
CDC against D-17 cells. As shown in Figure 2B, 134-mG2a-f
elicited a higher degree of CDC (28.8% cytotoxicity;p < 0.01) in D-17 cells compared with that elicited by control
mouse IgG2a (14.1% cytotoxicity) and the control PBS (8.8%
cytotoxicity). These results demonstrated that 134-mG2a-f
promoted significantly higher levels of ADCC and CDC
against dEGFR-expressing D-17 cells.
Antitumor activities of 134-mG2a-f in the mouse
xenografts of D-17 cells

In the D-17 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
D-17 cells. The tumor volume was measured on days 7, 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 18 ( p < 0.01), 21 ( p < 0.01), and 25( p < 0.01) compared with that of the control mouse IgG(Fig. 3A).
The administration of 134-mG2a-f resulted in a 32% reduction of tumor volume compared with that of the control
mouse IgG on day 25 postinjection. Furthermore, tumors
from the 134-mG2a-f-treated mice weighed significantly less

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FIG. 1. Flow cytometry and immunocytochemistry using recEMab-134 and 134-mG2a-f. (A, B) D-17 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) D-17 cells were incubated with buffer control, 10 mg/mL of recEMab-134, or 10 mg/mL of 134-mG2a-f for 1 hour atroom temperature. The cells were further incubated with Alexa Fluor 488-conjugated anti-mouse IgG and DAPI for
45 minutes at room temperature. Scale bars, 20 mm. (D) Determination of the binding affinity of 134-mG2a-f for D-17 cells
using flow cytometry. D-17 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.

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ANTITUMOR ACTIVITIES BY DEFUCOSYLATED ANTI-EGFR MAB

FIG. 2. Evaluation of ADCC and CDC elicited by
134-mG2a-f. (A) ADCC elicited by 134-mG2a-f, control
mouse IgG2a, or control PBS targeting D-17 cells. Asterisks
indicate statistical significance (*p < 0.05; Tukey’s test).
(B) CDC elicited by 134-mG2a-f, control mouse IgG2a, or
control PBS targeting D-17 cells. Values are mean – SEM.
Asterisks indicate statistical significance (**p < 0.01,*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.
than those from the control IgG-treated mice (29% reduction;p < 0.05, Fig. 3B). Tumors that were resected from mice on
day 25 are demonstrated in Figure 3C.
Total body weights did not differ significantly among thetwo groups (Fig. 4A). The body appearance of mice on day 25
is demonstrated in Figure 4B.
Taken together, these results indicate that the administration of 134-mG2a-f effectively reduced the growth of D-17
xenografts.
Discussion

Cancer is a common cause of death in dogs.(17,18) Amongthem, canine OS is a highly metastatic and intractable cancer,
with 80% of dogs with OS dying from lung metastasis.(10)Canine OS shares various clinical and molecular similarities
with human OS, making it a spontaneously occurring large
animal disease model that can be used to identify biomarkers
and develop treatments.(11) Therefore, developing therapeutic strategies for canine OS will improve not only the clinicalresponse rate of canine OS but also that of human OS.

5

FIG. 3. Evaluation of antitumor activity of 134-mG2a-f in
D-17 xenografts. (A) D-17 cells (5 · 106 cells) were injectedsubcutaneously into the left flank. On day 7, 100 mg of 134mG2a-f or control mouse IgG in 100 mL PBS was 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 mean – SEM. Asterisks indicate statistical significance (**p < 0.01; ANOVA and Sidak’s multiple comparisons test). (B) Tumors of D-17 xenografts were resectedfrom 134-mG2a-f and control mouse IgG groups. Tumorweight on day 25 was measured from excised xenografts.
Values are mean – SEM. Asterisk indicates statistical significance (*p < 0.05, Welch’s t-test). (C) Resected tumors of D-17
xenografts from the control mouse IgG and 134-mG2a-f groups
on day 25. Scale bar, 1 cm.

In canine OS, surgery (limb amputation or limb-sparing
surgery) is a first-line treatment. Chemotherapy including
carboplatin, cisplatin, and doxorubicin is also used for adjuvant and/or neoadjuvant therapy.(19–22) These treatments
have been shown to result in longer survival times than amputation alone.(23) However, these therapies significantly
reduce the canine quality of life. Therefore, it is essential toestablish a new therapeutic modality.
Because original antibody therapies for dogs have not been
established for most diseases including OS, antibody drugs
for humans have been used in their place. For example, thehEGFR and dEGFR sequences are 91% identical, and some

6

NANAMIYA ET AL.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by the Japan Agency
for Medical Research and Development (AMED) under grant
numbers JP21am0401013 (to Yukinari Kato) andJP21am0101078 (to Yukinari Kato), and by the Japan Society
for the Promotion of Science ( JSPS) Grants-in-Aid for Scientific Research (KAKENHI) under grant numbers
21K15523 (to Teizo Asano), 21K07168 (to Mika K. Kaneko), 20K16322 (to Masato Sano), and 19K07705 (to YukinariKato).

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/22. For personal use only.

References

FIG. 4. Body weights and appearance of the mice implanted with D-17 xenografts. (A) Body weights of mice
implanted with D-17 xenografts were recorded on days 7,
11, 14, 18, 21, and 25. (B) Body appearance of mice on day
25. Scale bar, 1 cm.

anti-hEGFR mAbs are effective against canine tumors thatoverexpress dEGFR in vitro and/or in vivo.(16,24) D-17 is acanine OS cell line commonly used for various studies.(25–29)
Since D-17 cells express EGFR,(30,31) Mantovani et al. applied an EGFR-tyrosine kinase inhibitor to suppress D-17 cellproliferation.(30)
However, there is no study to apply an anti-EGFR mAb to
EGFR-expressing canine OS cell lines. In the present study,
we demonstrated that 134-mG2a-f could recognize endogenous dEGFR by flow cytometric (Fig. 1A, B) and immunocytochemical analyses (Fig. 1C) in D-17 cells. The use of ananti-EGFR mAb for canine OS with high expression of
EGFR may also be effective for canine OS cases that have notresponded well to treatment in the past.
The most critical aim of the present study was to investigate the antitumor activity of 134-mG2a-f for endogenousdEGFR-expressing canine tumor cells. 134-mG2a-f demonstrated growth inhibition of endogenous dEGFR-expressing
D-17 cells without body weight loss and skin abnormality(Figs. 3 and 4) although the antitumor activity of 134-mG2a-f
for D-17 is weaker than that for CHO/dEGFR(16) due to thelow expression level of dEGFR in D-17 cells (Fig. 1). These
results suggest that 134-mG2a-f may be useful for an antibody
treatment regimen for dEGFR-expressing canine OS. Future
investigations are needed to test the antitumor effects of 134mG2a-f against spontaneously develop canine tumors.

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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: August 23, 2021
Accepted: January 11, 2022