cells
ArticleDefucosylated Mouse–Dog Chimeric Anti-EGFR Antibody
Exerts Antitumor Activities in Mouse Xenograft Models ofCanine Tumors
Guanjie Li 1 , Tomokazu Ohishi 2, * , Mika K. Kaneko 3 , Junko Takei 3 , Takuya Mizuno 4 , Manabu Kawada 2 ,Masaki Saito 1 , Hiroyuki Suzuki 1 and Yukinari Kato 1,3, *
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Citation: Li, G.; Ohishi, T.; Kaneko,
M.K.; Takei, J.; Mizuno, T.; Kawada,
M.; Saito, M.; Suzuki, H.; Kato, Y.
Defucosylated Mouse–Dog Chimeric
Anti-EGFR Antibody Exerts
Antitumor Activities in Mouse
Xenograft Models of Canine Tumors.
Cells 2021, 10, 3599. https://doi.org/
10.3390/cells10123599
Academic Editor: Silvia La Monica

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai, Miyagi 980-8575, Japan; clownair716@gmail.com (G.L.); saimasa@med.tohoku.ac.jp (M.S.);
hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.)
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
18-24 Miyamoto, Numazu-shi, Shizuoka 410-0301, Japan; kawadam@bikaken.or.jp
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan; k.mika@med.tohoku.ac.jp (M.K.K.);
lcdoosur@tmd.ac.jp (J.T.)
Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of Veterinary Medicine,
Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan; mizutaku@yamaguchi-u.ac.jp
Correspondence: ohishit@bikaken.or.jp (T.O.); yukinarikato@med.tohoku.ac.jp (Y.K.);
Tel.: +81-55-924-0601 (T.O.); +81-22-717-8207 (Y.K.)

Abstract: The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene
amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR)
monoclonal antibody, namely EMab-134, which detects hEGFR and dog EGFR (dEGFR) with highsensitivity and specificity. In this study, we produced a defucosylated mouse–dog chimeric antiEGFR monoclonal antibody, namely E134Bf. In vitro analysis revealed that E134Bf highly exerted
antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against a canine
osteosarcoma cell line (D-17) and a canine fibroblastic cell line (A-72), both of which express endogenous dEGFR. Moreover, in vivo administration of E134Bf significantly suppressed the developmentof D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that
E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable aspart of an antibody treatment regimen for dogs.
Keywords: EGFR; mouse–dog chimeric antibody; ADCC; CDC; canine osteosarcoma; antitumor activity

Received: 3 December 2021
Accepted: 17 December 2021
Published: 20 December 2021

1. Introduction
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine
kinases, which can form homo- or heterodimers with other EGFR family members, including HER2 (ErbB2/neu), HER3 (ErbB3), and HER4 (ErbB4) [1,2]. These dimers promote cellproliferation through the activation of several signaling pathways, such as the PI3K-AKTmTOR, Ras-Raf-MEK-ERK, and JAK-STAT pathways [3]. EGFR overexpression is observedin various tumors—including lung [4], breast [5], and colorectal carcinomas [6]; glioblastomas [7]; and osteosarcomas [8,9]—and contributes to tumor malignancy by augmenting
the aforementioned signaling pathways.
Osteosarcoma is the most common primary bone tumor in dogs and leads to metastasis [10]. Canine osteosarcoma has only a 45% 1-year survival rate, and its incidence is
27 times higher than that in humans [11]. The therapeutic measures for canine osteosarcoma include surgery, radiotherapy, and chemotherapy [12,13]. However, they are notsufficiently effective. Thus, new therapeutic strategies for canine osteosarcoma need to
be developed.

4.0/).

Cells 2021, 10, 3599. https://doi.org/10.3390/cells10123599

https://www.mdpi.com/journal/cells

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By immunizing mice with purified recombinant hEGFR ectodomain (hEGFRec) from
cultures of hEGFRec-overexpressed human glioblastoma, LN229, cells, we previously
developed a novel anti-human EGFR (hEGFR) monoclonal antibody (mAb), namely EMab134 [14]. EMab-134 can be used in Western blotting, flow cytometry, and immunohistochemistry. The mouse IgG2a version of EMab-134 (134-mG2a ) demonstrates antitumor activities
in mouse xenograft models of hEGFR-expressing 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]. The 134-mG2a -f exhibits ADCC
and complement-dependent cytotoxicity (CDC) in dog EGFR (dEGFR)-overexpressed
CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenograft models of
CHO/dEGFR cells [16]. In this study, we investigated the antitumor activities of a defucosylated mouse–dog anti-EGFR mAb (E134Bf) against D-17 and A-72 xenografts, both ofwhich endogenously express dEGFR.
2. Materials and Methods
2.1. Cell Lines
CHO-K1, a canine osteosarcoma cell line (D-17 [17]), and a canine fibroblast cell line
(A-72 [18]) were obtained from the American Type Culture Collection (Manassas, VA, USA).
CHO/dEGFR was established in our previous study [16]. CHO-K1 and CHO/dEGFR
were cultured in RPMI medium (Nacalai Tesque, Inc., Kyoto, Japan), D-17 in Minimum
Essential Medium (Nacalai Tesque, Inc., Kyoto, Japan), and A-72 in Dulbecco’s Modified
Eagle Medium (DMEM; Nacalai Tesque, Inc., Kyoto, Japan). Those media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc.,
Waltham, MA, USA), 1 mM of sodium pyruvate, 100 units/mL of penicillin, 100 µg/mL of
streptomycin, and 0.25 µg/mL of amphotericin B (Nacalai Tesque, Inc., Kyoto, Japan). The
cells were cultured at 37 ◦ C in a humidified atmosphere containing 5% CO2 . The negativity
for mycoplasma infection was confirmed using the TaKaRa Mycoplasma Detection Set(Takara Bio, Shiga, Japan).
2.2. Animals
All animal experiments were conducted according to relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. Animal experiments for
antitumor activity were approved by the Institutional Committee for Experiments of theInstitute of Microbial Chemistry (permit No. 2021-021). The mice were maintained in a
specific pathogen-free environment (23 ± 2 ◦ C, 55 ± 5% humidity) on an 11 h light/13 hdark cycle with food and water supplied ad libitum throughout the experimental period. In
addition, the mice were monitored for health and weight every 2–5 days during the 3-weekperiod for each experiment. We determined the loss of original body weight of more than
25% or a maximum tumor size greater than 3000 mm3 as humane endpoints for euthanasia.
The mice were euthanized by cervical dislocation and death was verified by respiratory
and cardiac arrest.
2.3. Antibodies
The anti-hEGFR mAb, EMab-134, was developed as previously described [14]. To
produce E134B, we subcloned the VH cDNA of EMab-134 and CH cDNA of dog IgGB
into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan),along with the VL cDNA of EMab-134 and CL cDNA of dog kappa light chain into thepCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation). Two vectors of E134B
were transfected into BINDS-09 cells (FUT8-deficient ExpiCHO-S cells) using the ExpiCHO Expression System (Thermo Fisher Scientific Inc., Waltham, MA, USA) [16]. The
resulting mAb, E134Bf, was purified with Protein G-Sepharose (GE Healthcare Biosciences,
Pittsburgh, PA, USA) [16]. Dog IgG was purchased from Jackson ImmunoResearch Inc.
(West Grove, PA, USA).

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2.4. Flow Cytometry
The D-17 and A-72 cells (1 × 105 cells/sample) were harvested by brief exposure to
0.25% trypsin in 1 mM ethylenediamine tetraacetic acid (EDTA, Nacalai Tesque, Inc.). Afterwashing with a blocking buffer of 0.1% bovine serum albumin (BSA) in phosphate-bufferedsaline (PBS), the cells were treated with 1 µg/mL of E134Bf or control blocking buffer for
30 min at 4 ◦ C. The cells were then incubated in FITC-conjugated anti-dog IgG at a dilutionof 1:1000 (Thermo Fisher Scientific Inc.) for 30 min at 4 ◦ C. Fluorescence data were collectedusing 488 nm laser and 525/50 bandpass filter of the EC800 Cell Analyzer (Sony Corp.,
Tokyo, Japan). No gating was used for the data analysis. All experiments were conductedin triplicate.
2.5. Determination of Binding Affinity
The D-17 and A-72 cells (1 × 105 cells/sample) were suspended in 100 µL of seriallydiluted E134Bf (0.006–100 µg/mL), followed by FITC-conjugated anti-dog IgG (1:200).
Fluorescence data were collected using 488 nm laser and 525/50 bandpass filter of theEC800 Cell Analyzer. No gating was used for the data analysis. All experiments wereconducted in triplicate. The dissociation constant (KD ) was calculated by fitting the binding
isotherms to built-in one-site binding models in GraphPad Prism 8 (GraphPad Software,Inc., La Jolla, CA, USA).
2.6. Immunocytochemistry
The D-17 and A-72 cells (1 × 105 cells/sample) were fixed with 4% paraformaldehyde
in PBS for 10 min and quenched with 50 mM NH4 Cl in PBS containing 0.2 mM Ca2+ and2 mM Mg2+ (PBSc/m) for 10 min. The cells were treated with blocking buffer (PBSc/m
supplemented with 0.5% BSA) for 30 min and incubated with 10 µg/mL of E134Bf or
the control blocking buffer for 1 h. The cells were then incubated with Alexa Fluor 488conjugated anti-dog IgG (1:400; Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA, USA) and DAPI (Thermo Fisher Scientific Inc.) for 45 min. Fluorescence images wereobtained using a BZ-X800 digital microscope (Keyence, Osaka, Japan) at a magnification of
40× with a GFP filter channel (green) and a DAPI filter channel (blue). All experiments
were conducted in triplicate.
2.7. ADCCADCC assay was performed as previously described [16,19–26]. Briefly, caninemononuclear cells derived from dog blood were obtained from Yamaguchi University andresuspended in DMEM with 10% FBS to be used as effector cells. Target cells (D-17 andA-72) were labeled with 10 µg/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 cells ratio, 50:1), 100 µg/mL of E134Bf
or the control dog IgG. After 4.5 h of incubation at 37 ◦ C, the release of calcein into thesupernatant was measured in each well. The fluorescence intensity was determined using
a microplate reader (PowerScan HT; BioTek Instruments, Inc., Winooski, VT, USA) withexcitation and emission wavelengths of 485 and 538 nm. Cytolytic activity (% lysis) was
calculated as follows: % lysis = (E − S)/(M − S) × 100, where E denotes the fluorescencemeasured of the combined cultures of the target and effector cells; S, the spontaneous
fluorescence of the target cells only; and M, 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 EDTA. All experiments were conducted in triplicate.
2.8. CDC
The CDC assay was performed as described previously [27–29]. Briefly, D-17 and A-72
cells were labeled with 10 µg/mL calcein AM and resuspended in the medium. They werethen 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, ON, Canada) andCells 2021, 10, 3599

4 of 16100 µg/mL of E134Bf or control dog IgG. After 4 h of incubation at 37 ◦ C, we measured
the release of calcein into the supernatant of each well. The fluorescence intensity wascalculated as described in Section 2.7. All experiments were conducted in triplicate.
2.9. Influence of E134Bf on EGF-Stimulated Cell GrowthA-72 cells (1 × 104 cells/well) were plated in each well of a 96-well plate. The cells were
either left untreated or treated with 1 ng/mL of canine recombinant EGF (SinoBiologicalInc., Beijing, China) with or without 20 µg/mL of normal dog IgG or E134Bf (n = 3 ineach group). After 36 h of incubation, cell growth was determined using the CellTiter
96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA),according to the manufacturer’s protocol.
2.10. Antitumor Activity of E134Bf in Xenografts of D-17 and A-72 Cells
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 old. D-17 or A-72 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, USA); 100 µL of this suspension (5 × 106 cells) was subcutaneously injected
into the left flank of the mice.
On day 7 post-inoculation, 100 µg of E134Bf (n = 8) or control dog IgG (n = 8) in 100 µL
PBS was intraperitoneally injected. Additional antibody inoculations were performed ondays 14 and 21. This regimen was selected based on prior studies [15,30]. Canine mononuclear cells, which were obtained from Yamaguchi University, were injected surrounding
the tumors on days 7, 14, and 21. At 25 days following cell implantation, all mice wereeuthanized by cervical dislocation. The tumor diameters and volumes were determined aspreviously described [16].
2.11. Immunohistochemistry
Immunohistochemistry was conducted as previously described [30]. Briefly, paraffinembedded xenograft tumor tissues were sectioned and placed on glass microscope slides.
After the sections were deparaffinized, they were boiled in buffered sodium citrate solution(0.01 mol/L, pH 6.0) for 10 min and subjected to immunohistochemical staining with
anti-Ki-67 antibody (ab15580, 1:1000; Abcam, Cambridge, UK), followed by horseradish
peroxidase-conjugated secondary antibody for rabbit IgG for 30 min. Then, the tissues
were stained with 3,30 -diaminobenzidine using the ChemMate EnVision Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The slides were briefly immersed in hematoxylin
for counterstaining and then observed under a Nikon Biophot microscope (Nikon, Tokyo,
Japan) and photographed using a digital camera (Nikon Digital Sight DS-Ri1, Nikon). The
photographs were taken under 400× magnification (Supplementary Figure S3A). Ki-67positive cells were counted from five randomly selected fields (Supplementary Figure S3B).
2.12. Statistical Analyses
All data are expressed as mean ± standard error of the mean (SEM). 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 andmouse weight. All calculations were performed using GraphPad Prism 8. A p-value of<0.05 was considered statistically significant.
3. Results
3.1. Flow Cytometry Analysis against D-17 and A-17 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 [16]. In this study, we
produced a defucosylated mouse–dog chimeric anti-EGFR mAb (E134Bf) by combining the
VH and VL of EMab-134 with the CH and CL of dog IgG, respectively (Figure 1). E134Bf also

Cells 2021, 10, 3599

5 of 16detected canine cell lines, such as D-17 and A-72 cells, although the endogenous6 dEGFR
Cells 2021, 10, x FOR PEER REVIEW
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expression level is lower than that of CHO/dEGFR cells (Figure 2A).

Figure1. Production
E134Bf(core-fucose-deficient(core-fucose-deficient dog
dog IgGB)
IgGB) from1).
Figure1. Production
ofofE134Bf
from EMab-134
EMab-134(mouse(mouseIgG
IgG
1 ).

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7 of 17

Figure 2. Flow cytometry using E134Bf. (A) CHO-K1, CHO/dEGFR, D-17, and A-72 cells were treated with E134Bf or
buffer control, followed by FITC-conjugated anti-dog IgG. (B) Determination of the binding affinity of E134Bf for CHO-K1,
CHO/dEGFR, D-17, and A-72 cells via flow cytometry. The cells were suspended in 100 µL of serially diluted E134Bf,
followed by the addition of FITC-conjugated anti-dog IgG. Fluorescence data were collected using the EC800 Cell Analyzer.

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Kinetic analysis of the interactions of E134Bf with the D-17 and A-72 cells was conducted via flow cytometry. As presented in Figure 2B, the KD for the interaction of E134Bf
with the D-17 and A-72 cells was 5.5 × 10−10 M and 6.0 × 10−10 M, respectively, indicatingthat E134Bf exhibits high affinity for D-17 and A-72 cells. On the contrary, the KD for theinteraction between the E134Bf and CHO/dEGFR cells was 3.2 × 10−9 M, indicating thatthe binding affinity of E134Bf for endogenous dEGFR in canine cancer cells is higher thanthat for exogenous dEGFR in CHO/dEGFR. Since dEGFR was not detected in CHO-K1
cells, we could not determine the binding affinity of E134Bf for CHO-K1 cells.
3.2. Immunocytochemistry against the D-17 and A-72 Cells Using E134Bf
Here, we investigated whether E134Bf was applicable for immunocytochemistry.
8 of 17
At first, we evaluated the specificity of E134Bf using the CHO-K1 and CHO/dEGFRcells. As a result, E134Bf detected dEGFR on the CHO/dEGFR cells but not the CHOK1 cells (Figure 3A,B). The buffer control detected no signal for both the CHO-K1 andFigure 2. Flow cytometry using E134Bf. (A) CHO-K1, CHO/dEGFR, D-17, and A-72 cells were treated with E134Bf or
CHO/dEGFR cells. Next, we examined the binding of E134Bf to endogenous dEGFR on
buffer control, followed by FITC-conjugated anti-dog IgG. (B) Determination of the binding affinity of E134Bf for CHOthe D-17 and A-72 cells and found that E134Bf specifically detected endogenous dEGFR
K1, CHO/dEGFR, D-17, and A-72 cells via flow cytometry. The cells were suspended in 100 μL of serially diluted E134Bf,(Figure3C,D). These
results
indicatethat E134Bf
recognizes
andCellexogenous
followed by the addition of FITC-conjugatedanti-dog
IgG.
Fluorescencedata werecollectedendogenous
using the EC800
AnadEGFR
inimmunocytochemistry.
lyzer.

Cells 2021, 10, x FOR PEER REVIEW

Figure3.3.Immunocytochemistry
Immunocytochemistryusing
usingE134Bf.
E134Bf.CHO-K1
CHO-K1(A),(A),CHO/dEGFR
CHO/dEGFR(B),(B),D-17
D-17(C),(C),andandA-72
A-72
Figure
(D)
cells
were
incubatedwithabuffercontrol
or
10
μg/mL
of
E134Bffor
1h,followedbyincubation(D) cells were incubated with a buffer control or 10 µg/mL of E134Bf for 1 h, followed by incubation
with Alexa Fluor 488-conjugated anti-dog IgG and DAPI for 45 min. Fluorescence images werewith Alexa Fluor 488-conjugated anti-dog IgG and DAPI for 45 min. Fluorescence images were taken
taken using a fluorescence microscope BZ-X800. Scale bars, 20 μm.
using a fluorescence microscope BZ-X800. Scale bars, 20 µm.

3.3. E134Bf-Mediated ADCC and CDC in the D-17 and A-72 Cells
We investigated whether E134Bf was capable of mediating ADCC against the D-17 andA-72 cells. As presented in Figure 4A, E134Bf showed ADCC (14.0% cytotoxicity) against

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D-17 cells more effectively than did the control dog IgG (4.7% cytotoxicity; p < 0.05) and thecontrol PBS (4.3% cytotoxicity; p < 0.05). E134Bf also showed ADCC (23.6% cytotoxicity)
against A-72 cells more effectively than did the control dog IgG (9.2% cytotoxicity; p < 0.05)
and the control PBS (7.4% cytotoxicity; p < 0.05). On the contrary, E134Bf did 9notshow
Cells 2021, 10, x FOR PEER REVIEW
of 17
ADCC against CHO-K1 cells, which are EGFR-negative cells (Supplementary Figure S1A).

Figure4. Evaluation
ADCCandand CDC
CDC elicited(A)(A)
ADCC
elicitedby E134Bf,
controlcontrol
dog IgG,or control
Figure4. Evaluation
ofofADCC
elicitedby
byE134Bf.
E134Bf.
ADCC
elicitedby E134Bf,
dog
IgG, orPBS
controltargeting the D-17 and A-72 cells. (B) CDC elicited by E134Bf, control dog IgG, or control PBS targeting the D-17 and A-72
PBS targeting the D-17 and A-72 cells. (B) CDC elicited by E134Bf, control dog IgG, or control PBS targeting the Dcells. The values are expressed as mean ± SEM. Asterisks indicate statistical significance (** p < 0.01; * p < 0.05; n.s., not
17 andA-72 cells. The values are expressed as mean ± SEM. Asterisks indicate statistical significance (** p < 0.01;
significant; Tukey’s post hoc test). ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cyto* p <toxicity.
0.05; n.s., not significant; Tukey’s post hoc test). ADCC, antibody-dependent cellular cytotoxicity; CDC, complementdependent cytotoxicity.
3.4. E134Bf Did Not Inhibit the EGF-Stimulated Cell Growth of A-72

We next determined whether E134Bf could inhibit the EGF-stimulated EGFR activation, which leads to cell growth. As presented in Supplementary Figure S2, we identified

and survival of A-72, E134Bf did not inhibit the cell growth of A-72, similar to the control
dog IgG. These results indicated that E134Bf did not inhibit the activation of EGFR in response to EGF stimulation.
Cells 2021, 10, 3599

3.5. Antitumor Activities of E134Bf in the Mouse Xenografts of D-17 and A-72 Cells

9 of 16

In the D-17 xenograft models, E134Bf (100 μg) and dog IgG (100 μg) were injected
intraperitoneally into mice on days 7, 14, and 21, following the injection of the D-17 cells.
The tumor
volumewas measured
days 7,could
11, 14,mediate18, 21, and25against
post-injection.
The adWe theninvestigatedwhetheronE134Bf
CDC
D-17 cells.
Asministrationof E134Bf
resultedelicitedin a significant
reductionin tumordays
presented
in Figure
4B, E134Bfa higher degreeof CDC(53.6%development
cytotoxicity) on
in D-1711 (pcompared< 0.05), 14with(p < 0.01),
18 (p < by0.01),(p < 0.01),
and(32.4%25 (p <cytotoxicity;0.01) comparedwith andthat
cellsthat elicitedthe 21
controldog IgG
p < 0.05)of thedog IgG
5A). The administrationof E134Bf
resulted
in a 37%
reduction
inthecontrol
PBS (Figure(27.7% cytotoxicity;p < 0.01). E134Bf
also eliciteda higherdegreeof CDCtumor cytotoxicity)
volume compared
the control
dog
IgG
on daypost-injection.
(53.2%
in the with
A-72 thatcells ofcomparedwiththatelicitedby25
controldog IgG (44.4%
In the A-72
thePBS
administrationof the antibody
treatmentmoncytotoxicity;p < xenograft
0.05) and models,the control(40.0% cytotoxicity;p < 0.01).
On theandcontrary,
itoring didof thewere performed
the same with theD-17
cells.
The adminE134Bf
nottumor
showvolume
CDC against
CHO-K1 (Supplementary
Figure
S1B).
These
results
istration of
E134Bfresulted
in a significant
reductionin tumor
development
onagainstdays 14the(p
indicated
that
E134Bfpromotedsignificantly
higher levelsof ADCCand CDCdEGFR-expressing
D-17< 0.01), 18 (p < 0.01),
21 and(p < A-72
0.01),cells.
and 25 (p < 0.01) compared with that of the dog IgG(Figure 5B). The administration of E134Bf resulted in a 50% reduction in tumor volume
3.4.
E134Bfwith
Did Not
Growth of A-72
compared
thatInhibitof the thedogEGF-Stimulated
IgG on day 25Cell
post-injection.

Wedetermined
whether
could
thedayEGF-stimulated
EGFR
Thenext
tumorsof D-17 that
wereE134Bf
resected
frominhibit
mice on25 are presented
in activaFigure
tion,
which
leads
to cell
growth.
As presented in
Supplementary
Figure S2,lesswe thanidentified
6A. TheD-17
tumorsfromthe E134Bf-treatedmice
weighed significantly
those
A-72
EGF-responsive
cell line.
Althoughthe additionof EGF
resultedin cell growthfrom asthean
controldog IgG-treatedmice(42% reduction;p < 0.01,
Figure6B). Consistentwith
andA-72,staining
E134Bf revealed
did not inhibit
the cellreduced
growth proliferationof A-72, similar
to thecontrolthesesurvival
results,of
Ki-67
a markedlyindex
in E134Bfdog
IgG.
These(Supplementary
results indicatedthat E134Bf
notinhibitthewereactivation
offromEGFR
intreatedtumorsFigureS3). Thedid
A-72
tumorsthat
resectedmice
responseto
EGF
stimulation.
on day 25 are demonstrated in Figure 6A. The A-72 tumors from the E134Bf-treated miceweighed significantly less than those from the dog IgG-treated mice (42% reduction; p <3.5. Antitumor Activities of E134Bf in the Mouse Xenografts of D-17 and A-72 Cells
0.05, Figure 6B).
In
the
D-17
xenograft
E134Bf(100 µg)
anddiddog(100 µg) were
The
total
body
weightsmodels,of the D-17
xenograft
mice
notIgG
significantly
differinjectedamongintraperitoneally
into micedays
7, 14,
and 21, following
thexenograft
injection miceof theonD-17
the two groups (Figure
7A).onThe
body
appearanceof the D-17
daycells.
25 is
The
tumor
volumewasmeasured
on
days
7,
11,
14,
18,
21,
and
25
post-injection.
The
presented in Figure 7B. In the same way, the total body weights of the A-72 xenograft miceadministrationof
E134Bfresultedinasignificant
reductionintumor
developmentondays
did not significantly differ among the two groups (Figure 7A). The body appearance of11< 0.05),14 (p <mice0.01),on
18day(p <250.01),
21 (p < 0.01), inand
25 (p 7B.
< 0.01) compared with thatthe(pA-72
xenograftis demonstrated
Figureof theTaken
dog IgG(Figure5A).
The
administrationof
E134Bfresulted
37% reduction
in
together, these results indicate that the administrationinofa E134Bf
effectively
tumor
volumecompared
with
that
of
the
control
dog
IgG
on
day
25
post-injection.
reduced the growth of D-17 and A-72 xenografts.

Figure 5. Evaluation of tumor volume in the D-17 and A-72 xenograft models. (A) D-17 cells (5 × 106 cells) were
subcutaneously injected into the left flank. On day 7, 100 µg of E134Bf (n = 8) or control dog IgG (n = 8) in 100 µL PBS was
intraperitoneally injected into the mice; additional antibodies were then injected on days 14 and 21. The tumor volume wasmeasured on days 7, 11, 14, 18, 21, and 25 after the injection. (B) The A-72 cells (5 × 106 cells) were subcutaneously injected
into the left flank. On day 7, 100 µg of E134Bf (n = 8) or control dog IgG (n = 8) in 100 µL PBS was intraperitoneally injected
into the 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. The values are expressed as mean ± SEM. Asterisks indicate statistical significance(** p < 0.01; * p < 0.05; n.s., not significant; ANOVA and Sidak’s multiple comparisons test).

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In the A-72 xenograft models, the administration of the antibody treatment andmonitoring of the tumor volume were performed the same with the D-17 cells. The
administration of E134Bf resulted in a significant reduction in tumor development on days
14
(p < 0.01), 18 (p < 0.01), 21 (p < 0.01), and 25 (p < 0.01) compared with that of the dog
IgG
Cells 2021, 10, x FOR PEER REVIEW
11 of 17
(Figure 5B). The administration of E134Bf resulted in a 50% reduction in tumor volume
compared with that of the dog IgG on day 25 post-injection.
The tumors of D-17 that were resected from mice on day 25 are presented in Figure 6A.
Figure 5. Evaluation of tumor
volumein the D-17
and
A-72
xenograft models.
D-17 cells(5 × 106 cells)
were
subcutaThe
D-17 tumors
fromthe
E134Bf-treatedmice(A)weighedsignificantly
less
than
those fromneously injected into the left
flank.
On
day
7,
100
μg
of
E134Bf(n=8)
orcontrol
dog
IgG
(n
=
8)
in
100
μL
PBS
was withthe control dog IgG-treated mice (42% reduction; p < 0.01, Figure 6B). Consistentintraperitoneally injected into the mice; additional antibodies were then injected on days 14 and 21. The tumor volume
these results, Ki-67 staining revealed a markedly reduced proliferation index in E134Bfwas measured on days 7, 11, 14, 18, 21, and 25 after the injection. (B) The A-72 cells (5 × 106 cells) were subcutaneously
treated tumors (Supplementary Figure S3). The A-72 tumors that were resected frominjected into the left flank. On day 7, 100 μg of E134Bf (n = 8) or control dog IgG (n = 8) in 100 μL PBS was intraperitoneally
mice antibodies
on day 25 wereare demonstrated
Figure6A.21.
The
A-72
tumorsfrom
E134Bf-treatedinjected into the mice; additional
then injected onindays
14 andThe
tumor
volumewasthemeasuredonmice
significantly
lessthose fromthe ±dog
IgG-treatedmice (42%reduction;days 7, 11, 14, 18, 21, and 25
afterweighed
the injection.
The values
arethanexpressed
as mean
SEM.
Asterisks indicate
statistical< 0.05,
6B).
significance (** p < 0.01; * p <p0.05;
n.s.,Figure
not significant;
ANOVA and Sidak’s multiple comparisons test).

FigureFigure 6.
6. Evaluation
Evaluation ofof tumor
tumor weightweight inin thethe D-17
D-17 andand A-72
A-72 xenograft
xenograft models.
models. The
The tumors
tumors ofof thethe D-17
D-17 andand A-72
A-72 xenografts
xenografts
were
resectedfromthecontroldog
IgG
andE134Bfgroups.
(A)
Appearanceofresectedtumorsofthewere resected from the control dog IgG and E134Bf groups. (A) Appearance of resected tumors of the D-17
D-17 andand A-72
A-72
xenografts from the control dog IgG and E134Bf groups on day 25. Scale bar, 1 cm. (B) The tumor weight on day 25 was
xenografts from the control dog IgG and E134Bf groups on day 25. Scale bar, 1 cm. (B) The tumor weight on day 25 wasmeasured from the excised D-17 and A-72 xenografts. The values are expressed as mean ± SEM. Asterisk indicates statismeasured from the excised D-17 and A-72 xenografts. The values are expressed as mean ± SEM. Asterisk indicates statistical
tical significance (** p < 0.01, *p < 0.05, Welch’s t-test).
significance (** p < 0.01, * p < 0.05, Welch’s t-test).

The total body weights of the D-17 xenograft mice did not significantly differ among
the two groups (Figure 7A). The body appearance of the D-17 xenograft mice on day 25 ispresented in Figure 7B. In the same way, the total body weights of the A-72 xenograft mice
did not significantly differ among the two groups (Figure 7A). The body appearance of the
A-72 xenograft mice on day 25 is demonstrated in Figure 7B.

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11 of 16
12 of 17

Figure7.7.Body
Bodyweights
weightsand
andappearance
appearance of
ofthe
themicemiceimplanted
implantedwith
withthetheD-17
D-17andandA-72
A-72xenografts.
xenografts.
Figure(A)
Body
weightsofmice
implanted
with
the
D-17
and
A-72
xenografts
wererecordedon days7,(A) Body weights of mice implanted with the D-17 and A-72 xenografts were recorded on days
7, 11,
11, 14, 18, 21, and 25 (n.s.: not significant). (B) Body appearance of the D-17 and A-72 xenograft mice
14, 18, 21, and 25 (n.s.: not significant). (B) Body appearance of the D-17 and A-72 xenograft mice onon day 25. Scale bar, 1 cm. n.s., not significant.
day 25. Scale bar, 1 cm. n.s., not significant.

4. Discussion
Taken together, these results indicate that the administration of E134Bf effectively

Amongcanineofcancers,
osteosarcoma
is a highly metastatic and intractable cancer,
reducedthe growthD-17 andA-72 xenografts.
and about 80% of dogs with osteosarcoma die from lung metastases [10,31,32]. Because
4.
Discussion
canineosteosarcoma shares various molecular and clinical similarities with human osteosarcoma,
can be used
for identifying
and cancer,
developing
Amongcanine
canineosteosarcoma
cancers, osteosarcoma
is a highly
metastaticbiomarkers
and intractableandtreatments
for
human
Therefore,
the developmentof therapeuticstratabout
80% ofdogs
withosteosarcoma
osteosarcoma[11].
die from
lung metastases[10,31,32].
Because canine
egies for canine
osteosarcoma
will alsoand
improvetheclinical response
rate ofhuman osteosteosarcoma
sharesvarious molecularclinicalsimilarities
with humanosteosarcoma,
osarcoma.
In canine canosteosarcoma,
is a biomarkers
first-line treatment.
Chemotherapy,
incanineosteosarcoma
be used forsurgery
identifying
and developing
treatments
cludingcarboplatin,
cisplatin,
doxorubicin,
is also used offortherapeutic
adjuvant and/or
neoadjufor
humanosteosarcoma[11]. and
Therefore,
the development
strategies
forcanineosteosarcoma
alsotreatments
improve theclinicalof humanosteosarcoma.
In
vant therapy[33–36].will
These
have
beenresponse
shown torate
resultin longer
survival times
canineosteosarcoma,
surgery
is a first-line
treatment.
Chemotherapy,often
includingcarboplatin,
than amputation
alone[37]. Because
the toxicity
of chemotherapy
causessevere adcisplatin,and doxorubicin,
is alsodiarrhea,
used foranorexia,
adjuvant andand/orneoadjuvant therapy
verse effects,
such as vomiting,myelosuppression,
which[33–36].
lead toThese
treatments
have been
to resulttimestothan
amputationa significant
reductionin theshown
canine’squalityinoflonger
life, itsurvival
is importantestablish
a new
alone
[37]. Because
the toxicity of chemotherapy often causes severe adverse effects, such as
therapeuticmodality.
vomiting,
diarrhea,
anorexia,
myelosuppression,lead to a tumor
significant
Caninefibrosarcoma
is aandmalignant,infiltrating,which
mesenchymal
that reduction
mainly afinthecanine’squality
of
life,
it
is
importanttoestablishanewtherapeuticmodality.
fects the oral cavity of medium to large dogs [38]. In addition to surgery and radiotherapy,
chemotherapy has been used as adjuvant treatment for oral fibrosarcoma. Doxorubicin isCells 2021, 10, 3599

12 of 16

Canine fibrosarcoma is a malignant, infiltrating, mesenchymal tumor that mainly affects the oral cavity of medium to large dogs [38]. In addition to surgery and radiotherapy,
chemotherapy has been used as adjuvant treatment for oral fibrosarcoma. Doxorubicin is
the most commonly administered drug in association with surgery and/or radiation. However, the ability to control local disease still represents the major challenge. Recently, theeffect of two tyrosine kinase inhibitors (TKI), imatinib (BCR-ABL inhibitor) and masitinib(c-KIT and PDGF inhibitor), on canine fibrosarcoma cells was investigated [39]. However,there are no reports on the use of anti-EGFR drugs, including TKI or antibody drugs, forthe treatment of canine oral fibrosarcoma.
Antibody therapies are successful against various diseases and generally well tolerated
in humans [40]. Original antibody therapies for dogs have not been established for mostdiseases, including osteosarcoma; therefore, antibody drugs for humans have been usedas alternatives. For example, several tumor antigens that have been used for targeted
therapies in human cancers are also identified in canine malignancies, including EGFR,
HER2, VEGFR2, CD20, podoplanin, PD-1, and PD-L1 [16,41–49]. Particularly, the hEGFR
and dEGFR amino acid sequences are 91% identical, and some anti-hEGFR mAbs are
effective against canine tumors that overexpress dEGFR in vitro and/or in vivo [16,50].
Although it has been reported that the aberrant expression of EGFR in human osteosarcoma is associated with poor response to chemotherapy, distant metastasis, andreduced survival time of patients [51,52], precise information on EGFR expression andits association with clinical features in canine osteosarcoma has not been elucidated. In
addition, the prognostic and clinicopathological significance of the EGFR expression ofcanine osteosarcoma and its relevance to EGFR-targeted drugs have not been fully elucidated. Further exploring these relationships could provide new insights into the efficacy of
EGFR-targeted therapies for both humans and dogs.
D-17 is a canine osteosarcoma cell line commonly used for various studies [53–57].
Since the D-17 cells express EGFR [58,59], Mantovani et al. applied an EGFR tyrosine
kinase inhibitor to suppress D-17 cell proliferation [58]. On the contrary, there has been no
study on the application of an anti-EGFR mAb to EGFR-expressing canine osteosarcoma
cell lines. In this study, we demonstrated that E134Bf could recognize the endogenousdEGFR of D-17 cells via flow cytometry and immunocytochemistry (Figures 2A and 3). The
most critical aim of the present study was to investigate the antitumor activity of E134Bf
for endogenous dEGFR-expressing canine tumor cells. E134Bf demonstrated growth
inhibition of endogenous dEGFR-expressing D-17 cells without body weight loss and skinabnormality (Figures 5–7).
A canine fibroblast cell line, A-72, was established from a tumor surgically removed
from a golden retriever and mainly used for virus research [18]. Although the histology ofthe original tumor has not been identified, the A-72 cells expressed EGFR (Figures 2 and 3),
and its growth was promoted by EGF (Supplementary Figure S2). Furthermore, E134Bf
could suppress xenograft growth (Figures 5 and 6), most likely through ADCC activity(Figure 4). Furthermore, through immunocytochemistry (Figure 3), the cytosolic dot-likestaining of dEGFR was preferentially observed in the A-72 cells, suggesting that the internalized dEGFR abundantly accumulates in the cytoplasm. Previously, we developed themouse–canine chimeric anti-dog podoplanin mAb P38B, conjugated with emtansine as thepayload, which demonstrated an antitumor effect against dog podoplanin -overexpressed
cells [60]. It would be interesting to examine the sensitivity of an E134Bf-drug conjugate to
the A-72 cells.
These results suggest that E134Bf may be useful for an antibody treatment regimen
for dEGFR-expressing canine tumors, which would lead to the establishment of EGFRtargeted immunotherapy. However, this study was limited by the number of canine tumor
samples. Future investigations are needed to test the antitumor effects of E134Bf against
spontaneously developed canine tumors and its efficacy compared to other caninizedmonoclonal antibodies.

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Supplementary Materials: The following are available online at https://www.mdpi.com/article/
10.3390/cells10123599/s1. Figure S1: Evaluation of ADCC and CDC by E134Bf against CHO-K1,
EGFR-negative cells elicited., Figure S2: E134Bf did not inhibit the cell growth of A-72 stimulated by
EGF., Figure S3: Ki-67 staining in the xenograft tumor tissues.
Author Contributions: Conceptualization, M.K. and Y.K.; methodology, M.K.K., T.M., and H.S.;
validation, J.T. and H.S.; investigation, G.L., T.O., and M.S.; resources, T.M.; writing, G.L. and T.O.;
funding acquisition, Y.K. All authors have read and agreed to the published version of the manuscript.
Funding: 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.) and by theJapan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI)
under grant numbers 21K07168 (to M.K.K.) and 19K07705 (to Y.K.).
Institutional Review Board Statement: All animal experiments were carried out in accordance withthe guidelines and regulation to minimize animal suffering and distress in the laboratory. Animal
experiments for antitumor activity were approved by the Institutional Committee for Experiments of
the Institute of Microbial Chemistry (permit No. 2021-021).
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets used and/or analyzed during the study are available
from the corresponding author on reasonable request.
Acknowledgments: We would like to thank Akiko Harakawa (Institute of Microbial Chemistry(BIKAKEN), Numazu, Microbial Chemistry Research Foundation) for technical assistance in the
animal experiment.
Conflicts of Interest: The authors declare no conflict of interest associated with this manuscript.

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