MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 1, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0023
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Antitumor Activities in Mouse Xenograft Models of Canine
Fibroblastic Tumor by Defucosylated Mouse-Dog Chimeric
Anti-HER2 Monoclonal Antibody (H77Bf)

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Hiroyuki Suzuki,1,* Teizo Asano,2,* Tomokazu Ohishi,3,4,* Takeo Yoshikawa,5 Hiroyoshi Suzuki,6Takuya Mizuno,7 Tomohiro Tanaka,2 Manabu Kawada,4 Mika K. Kaneko,2 and Yukinari Kato1,2,5

Human epidermal growth factor receptor 2 (HER2) is a cell surface type I transmembrane glycoprotein that isoverexpressed on a variety of solid tumors and transduces the oncogenic signaling upon homo- and heterodimerization with HER families. Anti-HER2 monoclonal antibodies (mAbs) including trastuzumab and its
antibody-drug conjugate have been shown to improve patients’ survival in HER2-positive breast, gastric, andlung cancers. Canine tumors have advantages as naturally occurring tumor models, and share biological andhistological characteristics with human tumors. In this study, we generated a defucosylated version of mousedog chimeric anti-HER2 mAb (H77Bf) derived from H2Mab-77 (mouse IgG1, kappa). H77Bf possesses thehigh binding affinity (a dissociation constant: 8.7 · 10-10 M) for a dog HER2 (dHER2)-expressing canine
fibroblastic tumor cell line (A-72). H77Bf exhibited antibody-dependent cellular cytotoxicity and complementdependent cytotoxicity for A-72 cells. Moreover, intraperitoneal administration of H77Bf significantly suppressed the development of A-72 tumor compared with the control dog IgG in a mouse xenograft model. These
results indicate that H77Bf exerts antitumor activities against dHER2-expressing canine cancers, which could
provide a valuable information for canine cancer treatment.
Keywords: HER2, mouse-dog chimeric antibody, antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, canine tumor, antitumor activity

gests the involvement of immunologic engagement in antibody therapy.5
With the increase in lifespan of both humans and dogs,
the increased cancer incidence has been observed as well. In
canine tumors, the overexpression of dog HER2 (dHER2)
has been reported in 32% of canine mammary tumors,6 81%
of intestinal tumor, 42% of rectal carcinomas, and 28%
of cutaneous squamous cell carcinomas7 in accordancewith the American Society of Clinical Oncology and theCollege of American Pathologists guidelines for HER2
immunostaining.

IntroductionHuman epidermal growth factor receptor 2 (HER2)
overexpression is approximately observed in 20%–
30% of human breast cancers, which are associated with poor
prognosis and higher rates of recurrence.1 In 1998, trastuzumab was first approved by the U.S. Food and Drug Administration for treatment of HER2-positive breast cancers2
and later in HER2-positive gastric cancers.3 Trastuzumab has
been the most effective therapy for HER2-positive breast
cancer for more than 20 years.4 Clinically, the efficacy sug-

Departments of 1Molecular Pharmacology and 2Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Japan.
3
Microbial Chemistry Research Foundation, Institute of Microbial Chemistry (BIKAKEN), Numazu-shi, Japan.
4
Laboratory of Oncology, Microbial Chemistry Research Foundation, Institute of Microbial Chemistry (BIKAKEN), Shinagawa-ku, Japan.
5
Department of Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
6
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
7
Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of Veterinary Medicine, Yamaguchi University,
Yamaguchi, Japan.
*These authors contributed equally to this study.

34

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DEFUCOSYLATED ANTI-HER2 MAB

In canine tumors, the initiation, disease progression, genemutations, growth factors, and immune responses are similar to
those of human cancers.8 Doyle et al demonstrated the vaccineinduced dog epidermal growth factor receptor (dEGFR)/dHER2-specific immunity in spontaneous canine cancer.9
Dogs with EGFR-positive tumors were immunized with apeptide of the dEGFR extracellular domain highly homologous
to dHER2. Serum analyses showed high titers of dEGFR/dHER2-binding antibodies with biological activity similar tothat of cetuximab and trastuzumab. The antibodies inhibited
EGFR intracellular signaling and tumor growth. These results
support the approach of antitumor immunity to dEGFR/dHER2
that could be relevant in combination with other immune
therapies including immune checkpoint inhibitors.
Previously, we developed anti-HER2 monoclonal antibodies (mAbs), such as H2Mab-19,10–13 H2Mab-41,14,15
H2Mab-77 (mouse IgG1, kappa),16 H2Mab-119,17 H2Mab139,18 and H2Mab-181,19,20 which detect not only HER2 but
also dHER2. In this study, we produced a defucosylatedmouse-dog chimeric anti-HER2 mAb (H77Bf) from H2Mab77, and investigated the ability of H77Bf to induce the antitumor efficacy against dHER2-positive tumors.
Materials and Methods
Cell lines

A canine fibroblastic cell line (A-72) was obtained fromthe American Type Culture Collection (Manassas, VA).
A-72 was cultured in Dulbecco’s modified Eagle medium(DMEM; 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, and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.). The cell lines weremaintained at 37C in a humidified atmosphere under 5%
CO2.
Animals

All animal experiments were performed following relevant
guidelines and regulations to minimize animal suffering anddistress in the laboratory. Animal experiments for antitumor
activity were approved by the Institutional Committee forExperiments of the Institute of Microbial Chemistry (Permit
No. 2021-056). Mice were maintained in a specific pathogenfree environment (23C – 2C, 55% – 5% humidity) on an 11hours 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. We determined the loss of original
body weight to a point >25% and/or a maximum tumor size
>2000 mm3 as humane endpoints for euthanasia. Mice wereeuthanized by cervical dislocation; death was verified byrespiratory and cardiac arrest.

35

was transfected into BINDS-09 cells (FUT8-deficient
ExpiCHO-S cells) using the ExpiCHO Expression System(Thermo Fisher Scientific, Inc.). The resulting mAb, H77Bf,
was purified with Ab-Capcher (ProteNova, Kagawa, Japan).
Dog IgG was purchased from Jackson ImmunoResearch, Inc.
(West Grove, PA).
Flow cytometric analysis

A-72 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 H77Bf or control blocking
buffer for 30 min at 4C. Then, cells were incubated in the
fluorescein isothiocyanate (FITC)-conjugated anti-dog IgG
at a dilution of 1:1000 (Thermo Fisher Scientific, Inc.) for
30 min at 4C. Fluorescence data were collected using theEC800 Cell Analyzer (Sony Corp., Tokyo, Japan).
Determination of binding affinity of H77Bf to A-72

A-72 cells were suspended in 100 lL of serially diluted
H77Bf (0.006–25 lg/mL) followed by the 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).
Antibody-dependent cellular cytotoxicity activityof H77Bf

Canine mononuclear cells (MNCs) were obtained from
Yamaguchi University and resuspended in DMEM with
10% FBS to be used as effector cells.21 Target cells werelabeled with 10 lg/mL Calcein AM (Thermo Fisher Scientific, Inc.) and resuspended in the same medium.22 Thetarget 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 H77Bf or control dog IgG. After 4.5 hours
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 wavelengthof 538 nm.
Cytolytic activity (% lysis) was calculated as follows:% lysis = (E - S)/(M - S) · 100, where ‘‘E’’ is the fluorescence measured in combined cultures of target and effector
cells, ‘‘S’’ is the spontaneous fluorescence of target cells
only, and ‘‘M’’ is the maximum fluorescence measured after
the lysis of all cells with a buffer containing 0.5% Triton
X-100, 10 mM Tris-HCl (pH 7.4), and 10 mM of EDTA.
Complement-dependent cytotoxicity of H77Bf

Antibodies

To generate H77B, we subcloned VH cDNA of H2Mab-77
and CH of dog IgGB into the pCAG-Ble vector (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan) along withVL cDNA of H2Mab-77 and CL cDNA of dog kappa lightchain into the pCAG-Neo vector (FUJIFILM Wako Pure
Chemical Corporation), respectively. The vector of H77B

Cells were labeled with 10 lg/mL Calcein AM and resuspended in the medium.23 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, Ontario, Canada) and 100 lg/mL of
H77Bf or control dog IgG. After 4.5 hours of incubation at
37C, we measured Calcein release into the supernatant forDownloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

36

SUZUKI ET AL.

FIG. 1. Flow cytometry using H77Bf. (A) A-72 cells were treated with 10, 1, 0.1, and 0.01 mg/mL of H77Bf (red lines) or
buffer control (black lines), followed by the treatment with FITC-conjugated anti-dog IgG. (B) Kinetic analysis of H77Bf
for A-72 cells using flow cytometry. A-72 cells were suspended in 100 mL of H77Bf, followed by the treatment with FITCconjugated anti-dog IgG. FITC, fluorescein isothiocyanate.
each well. Fluorescence intensity was calculated as describedin the Antibody-Dependent Cellular Cytotoxicity Activity(ADCC) of H77Bf section earlier.

Results

Antitumor activity of H77Bf in xenograftsof the A-72 cells

In previous study, we established an anti-HER2 mAb(H2Mab-77) using cancer-specific mAb method.16 In thisstudy, we produced a defucosylated mouse-dog chimericanti-HER2 mAb (H77Bf) by combining VH and VL of
H2Mab-77 with CH and CL of dog IgG, respectively. H77Bf
reacted with a canine cell line, A-72 cells in a dose-dependent
manner (Fig. 1A).

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 8 weeks of age. The 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); 100 lL of this suspension (5 · 106 cells) was
injected subcutaneously into the left flanks of the mice.
On day 8 postinoculation, 100 lg of H77Bf (n = 8) orcontrol dog IgG (n = 8) in 100 mL PBS were injected intraperitoneally. Additional antibody inoculations were performed on days 14 and 21. MNCs were injected surrounding
the tumors on days 8, 14, and 21. The tumor volume wasmeasured on days 7, 10, 14, 17, 21, 24, and 28 after the
injection of cells. At day 28 after cell implantation, all mice
were euthanized by cervical dislocation. Tumor diametersand volumes were determined as previously described.24

Flow cytometric analysis against the A-72 cells
using H77Bf

Statistical analyses

All data are expressed as mean – standard error of themean. Statistical analysis was conducted with Welch’s ttest for ADCC, complement-dependent cytotoxicity(CDC), and 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.

FIG. 2. Evaluation of ADCC and CDC elicited by H77Bf.
ADCC (left panel) and CDC (right panel) elicited by H77Bf
or control dog IgG targeting A-72 cells were evaluated. The
values are expressed as mean – SEM. Asterisks indicate
statistical significance (*p < 0.05). ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent
cytotoxicity; SEM, standard error of the mean .

DEFUCOSYLATED ANTI-HER2 MAB

Next, we measured the dissociation constant (KD) between
H77Bf and the A-72 cells using flow cytometry. As a result,
the KD was determined to be 8.7 · 10-10 M, indicating that
H77Bf exhibits high affinity for A-72 cells (Fig. 1B).
ADCC and CDC activities mediated by H77Bf against
the A-72 cells

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We next measured the ADCC and CDC activities of
H77Bf against the A-72 cells. H77Bf showed ADCC activity
against A-72 cells (23.7% cytotoxicity) more effectively
compared with the control dog IgG (6.32% cytotoxicity;p < 0.05) as shown in Figure 2 (left). H77Bf also showed
CDC activity against A-72 cells (75.0% cytotoxicity) more
effectively compared with the control dog IgG (56.5% cytotoxicity; p < 0.05) as shown in Figure 2 (right). These results indicated that H77Bf mediated significantly higher
levels of ADCC and CDC against A-72 cells.
Antitumor activities of H77Bf in the mouse xenograftsof A-72 cells

H77Bf (100 lg) or control dog IgG (100 lg) were injected
intraperitoneally into mice on days 8, 14, and 21, after the
A-72 cell inoculation. The tumor volume was measured on

37days 7, 10, 14, 17, 21, 24, and 28 after the A-72 cell inoculation. Administration of H77Bf induced the significant reduction of tumor volume on day 10 ( p < 0.05), 14 ( p < 0.01),
17 ( p < 0.01), 21 ( p < 0.01), 24 ( p < 0.01), and 28 ( p < 0.01)
compared with that of the control dog IgG administration(Fig. 3A). The administration of H77Bf resulted in a 52.3%
tumor volume reduction compared with that of the control
dog IgG on day 28 postinoculation.
The A-72 tumors were resected from mice on day 28, andtheir weights were measured. As a result, the tumor weight of
H77Bf-treated mice was significantly less than that of control
dog IgG-treated mice (57.8% reduction; p < 0.01) (Fig. 3B, C).
Total body weight of the A-72 xenograft mice did not
significantly differ among the two groups (Fig. 4A). The body
appearance of the A-72 xenograft mice on day 28 is presented
in Figure 4B.
Taken together, these results indicate that the administrationof H77Bf effectively reduced the growth of A-72 xenograft.
Discussion

Among canine IgG subclasses (A, B, C, and D), the B and
D subclasses are involved in ADCC.25 In this study, we
converted mouse IgG1 into type B of dog IgG, and produced adefucosylated mAb, which has been proved to exhibit more

FIG. 3. Evaluation of antitumor activity of H77Bf in A-72 xenograft models. (A) Evaluation of tumor volume of A-72
xenograft. A-72 cells (5 · 106 cells) were subcutaneously inoculated into the left flank. On day 8, 100 mg of H77Bf (n = 8) orcontrol dog IgG (n = 8) in 100 mL PBS was intraperitoneally injected into the mice; additional antibodies were injected ondays 14 and 21. The tumor volume was measured on days 7, 10, 14, 17, 21, 24, and 28 after the A-72 cell inoculation.
Asterisk indicates statistical significance (**p < 0.01, *p < 0.05). (B) Evaluation of tumor weight in the A-72 xenograftmodel. The tumor weight on day 28 was measured from the excised A-72 xenograft. The values are expressed asmean – SEM. Asterisk indicates statistical significance (**p < 0.01). (C) Appearance of resected tumors of the A-72 xenografts. The tumors of the A-72 xenograft were resected from the H77Bf and control dog IgG groups on day 28. Scale bar,
1 cm. PBS, phosphate-buffered saline.

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38

SUZUKI ET AL.

FIG. 4. Body weights and appearance of the mice inoculated with A-72 xenograft. (A) Body weights of the miceinoculated with the A-72 xenograft. Body weights of mice inoculated with the A-72 xenograft were recorded on days 7, 10,
14, 17, 21, 24, and 28 after the A-72 cell inoculation (n.s., not significant). (B) Body appearance of the mice inoculated with
the A-72 xenograft on day 28. Scale bar, 1 cm.

potent ADCC activity through binding to FcgRIIIa on NK
cells.26 We first confirmed the cross-reactivity of H77Bf to
A-72 cells (Fig. 1A), and found that H77Bf possesses comparable high binding affinity to A-72 cells (8.7 · 10-10 M,
Fig. 1B), compared with human breast cancer SK-BR-3 cells(7.3 · 10-9 M, determined by H2Mab-77) as reported previously.16 The quantitative analysis of affinity is important toapply an anti-human molecule mAb to dogs.
The H77Bf administration with canine MNCs resulted insignificant growth suppression in A-72 tumor (Fig. 3). This
result provided the evidence of H77Bf as a promising antibody therapy against dHER2-positive tumors. We also
showed the increased ADCC and CDC activities by H77Bf
with canine MNCs and complement, respectively (Fig. 2).
These results suggest that ADCC and CDC activity could be
involved in the antitumor activity by H77Bf. In the future
study, the evaluation of H77Bf against spontaneously developed dHER2-positive canine tumors will be required.
A canine fibroblastic tumor cell line, A-72, was establishedfrom a tumor surgically removed from a golden retriever andmainly used for virus research.27 Although the histology ofthe original tumor has not been identified, the A-72 cells
expressed both dHER2 (Fig. 1) and dEGFR.22 Furthermore,
the cytosolic dot-like staining of dEGFR was observed in the
A-72 cells, suggesting that the internalized dEGFR accumulates in the cytoplasm.22 Therefore, internalization of
HER2 by H77B should be evaluated for the development ofantibody-drug conjugate. Trastuzumab deruxtecan (T-DXd,
DS-8201) is a HER2-targeting antibody conjugated with anovel DNA topoisomerase I inhibitor.28
T-DXd showed promising clinical outcomes in metastatic
breast cancer.29 Currently, the clinical efficacy and safety of
T-DXd have been evaluated in various tumors. T-DXd has
been approved in not only HER2-positive breast cancers,29–31
but also HER2-mutant lung cancers.30 We previously generated a mouse-canine chimeric mAb against dog
podoplanin32–34 (P38B) conjugated with emtansine as thepayload (P38B-DM1).35 P38B-DM1 showed cytotoxicity to
podoplanin-positive cells and exhibited potent antitumor
activity than P38B in the xenograft model.35

Immunohistochemistry (IHC) is useful as a diagnostic tool
for the identification of neoplasms with histopathology. IHC
is routinely used to assist with the diagnosis and to determine
the specific treatment (e.g., trastuzumab) for human cancer
patients. Although IHC is not routinely used in canine tumors, growing number of studies have been looking for reliable diagnostic and/or prognostic IHC biomarkers including
dHER2.36 Therefore, the standardization of dHER2 IHC isnecessary similar to human breast cancer diagnosis. Our established H2Mab-77 mAb can be used for IHC,16 and its
caninized H77Bf exerts the antitumor activity against dHER2
positive cells (Fig. 3); therefore, the combination of H2Mab77 and H77Bf could be applied for both diagnosis and therapy for dHER2-expressed canine tumors.
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 JP22ama121008 (to Y.K.), JP22am0401013(to Y.K.), JP22bm1004001 (to Y.K.), JP22ck0106730(to Y.K.), and JP21am0101078 (to Y.K.).
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SUZUKI ET AL.

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doi: 10.1186/s13028-018-0417-1

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: yukinari.kato.e6@tohoku.ac.jp
Received: June 1, 2022
Accepted: September 30, 2022