ONCOLOGY REPORTS 48: 154, 2022

Defucosylated mouse‑dog chimeric anti‑HER2
monoclonal antibody exerts antitumor activities inmouse xenograft models of canine tumors
HIROYUKI SUZUKI1,  TOMOKAZU OHISHI2,  TEIZO ASANO3, TOMOHIRO TANAKA3, MASAKI SAITO1,TAKUYA MIZUNO4, TAKEO YOSHIKAWA5,  MANABU KAWADA2, MIKA K. KANEKO3 and  YUKINARI KATO1,3,5
1Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980‑8575;
2
Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Research Foundation,
Numazu, Shizuoka 410‑0301; 3Department of Antibody Drug Development, Tohoku University Graduate School of
Medicine, Sendai, Miyagi 980‑8575; 4Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of
Veterinary Medicine, Yamaguchi University, Yamaguchi 753‑8515; 5Department of Pharmacology,
Tohoku University Graduate School of Medicine, Sendai, Miyagi 980‑8575, Japan
Received April 15, 2022;  Accepted June 15, 2022
DOI: 10.3892/or.2022.8366

Abstract. Human epidermal growth factor receptor 2 (HER2)
overexpression has been reported in various types of cancer,
including breast, gastric, lung, colorectal and pancreaticcancer. A humanized anti‑HER2 monoclonal antibody (mAb),
trastuzumab, has been shown to improve survival of patients in
HER2‑positive breast and gastric cancer. An anti‑HER2 mAb,
H 2Mab‑77 (mouse IgG1, kappa) was previously developed.
In the present study, a defucosylated version of mouse‑dog
chimeric anti‑HER2 mAb (H77Bf) was generated. H77Bf
possesses a high binding‑affinity [a dissociation constant(KD): 7.5x10‑10 M, as determined by flow cytometric analysis]for dog HER2‑overexpressed CHO‑K1 (CHO/dHER2)
cells. H77Bf highly exerted antibody‑dependent cellularcytotoxicity (ADCC) and complement‑dependent cytotoxicity(CDC) for CHO/dHER2 cells by canine mononuclear cells andcomplement, respectively. Moreover, administration of H77Bf
significantly suppressed the development of CHO/dHER2
xenograft tumor in mice compared with the control dog IgG.
H77Bf also possesses a high binding‑affinity (KD: 7.2x10‑10 M)
for a canine mammary gland tumor cell line (SNP), andshowed high ADCC and CDC activities for SNP cells.
Intraperitoneal administration of H77Bf in mouse xenograftmodels of SNP significantly suppressed the development of
SNP xenograft tumors compared with the control dog IgG.
These results indicated that H77Bf exerts antitumor activities
against dHER2‑positive canine cancers, and could be valuable
treatment regimen for canine cancers.
Introduction

Correspondence to: Dr Hiroyuki Suzuki or Dr Yukinari Kato,

Department of Molecular Pharmacology, Tohoku University
Graduate School of Medicine, 2‑1 Seiryo‑machi, Aoba‑ku, Sendai,
Miyagi 980‑8575, JapanE‑mail: hiroyuki.suzuki.b4@tohoku.ac.jp
E‑mail: yukinari‑k@bea.hi‑ho.ne.jp; yukinari.kato.e6@tohoku.ac.jp

Abbreviations: HER2, human epidermal growth factor receptor 2;

mAb, monoclonal antibody; ADCC, antibody‑dependent cellularcytotoxicity; CDC, complement‑dependent cytotoxicity; FDA, Foodand Drug Administration; PI3K, phosphatidylinositol‑3 kinase; TKI,
tyrosine kinase inhibitor; FcγR, Fcγ receptor; NK, natural killer;
CMT, canine mammary tumor; RPMI, Roswell Park Memorial
Institute; PBS, phosphate‑buffered saline; KD, dissociation constant;
DAPI, 4',6‑diamidino‑2‑phenylindole; MNC, mononuclear cell;
SEM, standard error of the mean; IHC, immunohistochemistry;
T‑DXd, Trastuzumab deruxtecan

Key words: HER2, monoclonal antibody, ADCC, CDC, antitumor

activity

Human epidermal growth factor receptor 2 (HER2, also
known as ERBB2) is a cell surface type I transmembrane
glycoprotein that is highly expressed on various solid tumors
and enable a broad repertoire of oncogenic signaling uponhomo‑ and heterodimerization with HER/ERBB families.
HER2 overexpression is observed in ~20‑30% of humanbreast cancers, which are associated with poor prognosis andhigher rates of recurrence (1). In 1998, trastuzumab became
the first monoclonal antibody (mAb), which U.S. Food andDrug Administration (FDA) approved for treatment of
HER2‑positive breast cancers (2) and later in HER2‑positive
gastric cancers (3).
Trastuzumab was initially considered to inhibit HER2
signaling (4,5). Numerous studies have confirmed the inhibi‑tion of downstream phosphatidylinositol‑3 kinase (PI3K)/Akt
pathway, and the suppression of tumor cell proliferation (6‑8).
Concurrently, the HER2‑selective tyrosine kinase inhibi‑
tors (TKIs) such as lapatinib, neratinib and tucatinib, weredeveloped and exhibited a superior activity to suppress HER2
signaling (6,9,10). However, regardless of a weaker inhibitory

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SUZUKI et al:  DEFUCOSYLATED MOUSE‑DOG CHIMERIC ANTI‑HER2 mAb

activity to HER2 signaling, trastuzumab has exhibited greater
clinical efficacy than TKIs. Trastuzumab has been the mosteffective therapy for HER2‑positive breast cancer for morethan 20 years (11). Clinically, this difference in efficacy
suggests the involvement of immunologic engagement ofantibody therapy, hardly observed in TKIs (12).
Trastuzumab possesses an Fc domain which allows forthe direct engagement with Fcγ receptors (FcγRs) on various
types of immune cells. The Fc γR engagement allows forphagocytic engulfment of antibody‑bound pathogens orcells, termed antibody‑dependent cellular phagocytosis. The
FcγR‑mediated signaling activates dendritic cells, macro‑phages and neutrophils, which can alter adaptive immune
responses through antigen presentation, cytokine production
and chemotaxis. Furthermore, the Fc γR engagement canstimulate natural killer (NK) cells which attack and lyse thetarget cells, termed antibody‑dependent cellular cytotoxicity(ADCC) (13). Margetuximab contains several optimization
mutations and exhibits improved Fc γRIIIA engagement
and ADCC activity compared with the parental Ab trastu‑
zumab (14). Margetuximab was recently approved by FDA
in heavily pretreated patients based on modest but significant
improvement in progression‑free survival (15,16). Moreover,the Fc domain can trigger the activation of complement
family, and exert the complement‑dependent cytotoxicity(CDC) (17,18).
With the increase in lifespan of both humans and dogs,
the increased cancer incidence has been observed as well.
Mammary neoplasia is the most frequently observed in dog
tumors (19). Among them, ~50% are malignant. These spon‑taneous canine mammary tumors (CMT) share biological andhistological characteristics with human breast carcinoma (20).
Compared with murine model, CMT models have advantages
as a naturally occurring models of human cancers (21). In
canine tumors, the overexpression of dog HER2 (dHER2) has
been reported not only in mammary carcinoma (22) but also
osteosarcoma (23), bladder carcinoma (24), and anal sac gland
carcinoma (25). Furthermore, in accordance with the American
Society of Clinical Oncology and the College of American
Pathologists guidelines for HER2 immunostaining, dHER2
has been revealed to be overexpressed in 32% of CMT (26),
81% of intestinal tumor, 42% of rectal carcinomas, and 28%
of cutaneous squamous cell carcinomas (27). Additionally, aHER2‑expressed recombinant Listeria vaccine administration
resulted in the induction of anti‑dHER2 immunity, which
resulted in the reduced incidences of metastasis, and prolonged
survival in a phase I study for canine osteosarcoma (28). These
clinical outcomes promoted the evaluation of anti‑dHER2
mAbs as a therapeutic modality for canine cancers.
Previously, an anti‑HER2 mAb, H 2Mab‑77 (mouse IgG1,
kappa), was developed (29). In the present study, a defucosyl‑
ated mouse‑dog chimeric anti‑HER2 mAb (H77Bf) was
produced. The present study aimed to investigate the abilityof H77Bf to induce ADCC, CDC and antitumor efficacy indHER2‑expressing cells.
Materials and methods
Cell lines. A canine mammary gland tumor cell line, SNP,
was purchased from the Cell Resource Center for Biomedical

Research Institute of Development, Aging and Cancer at
Tohoku University (Miyagi, Japan) (30). CHO‑K1 cells werepurchased from the American Type Culture Collection.
Dog HER2 (accession no. NM_001003217)‑overexpressed
CHO‑K1 (CHO/dHER2) was established by transfection ofpCAG/3xRIEDL‑dHER2 into CHO‑K1 cells as previously
described (31). 3xRIEDL sequence represented three repeatof RIEDL amino acid sequence (32). RIEDL tag is an affinity
tag that is used for the one‑step membrane protein purifica‑tion (32‑36). CHO‑K1, CHO/dHER2, and SNP were cultured
in RPMI‑1640 medium (Nacalai Tesque, Inc.), supplemented
with 10% heat‑inactivated fetal bovine serum (FBS; Thermo
Fisher Scientific, Inc.), 100 µg/ml streptomycin, 100 units/mlof penicillin, and 0.25 µg/ml amphotericin B (Nacalai Tesque,Inc.). The cell lines were maintained at 37˚C in a humidified
atmosphere under 5% CO2.
Animals. Animal experiments were performed following
regulations and guidelines to minimize animal distress andsuffering in the laboratory. Animal experiments for antitumor
activity of H77Bf were approved (approval no. 2021‑056) by
the Institutional Committee for Experiments of the Institute ofMicrobial Chemistry (Numazu, Japan). Mice were maintainedon an 11 h light/13 h dark cycle with food and water supplied
ad libitum in a specific pathogen‑free environment across theexperimental period. Mice were monitored for weight andhealth every 2‑5 days during the experiments. The loss oforiginal body weight was determined to a point >25% and/or
a maximum tumor size >3,000 mm3 as humane endpoints foreuthanasia.
Antibodies. Anti‑HER2 mAb H 2 Mab‑77 was establishedas previously described (29). To generate H77B, we
subcloned V H cDNA of H 2Mab‑77 and CH of dog IgGB into
the pCAG‑Ble vector (FUJIFILM Wako Pure Chemical
Corporation). V L cDNA of H 2Mab‑77 and CL cDNA of dog
kappa light chain were also subcloned into the pCAG‑Neo
vector (FUJIFILM Wako Pure Chemical Corporation).
The vector of H77B was transduced into BINDS‑09(FUT8‑deficient ExpiCHO‑S) cells using the ExpiCHO
Expression System (Thermo Fisher Scientific, Inc.) (37‑41).
H77Bf was purified using Ab‑Capcher (ProteNova Co., Ltd.).
Dog IgG was purchased from Jackson ImmunoResearchLaboratories, Inc.
Flow cytometry. CHO‑K1, CHO/dHER2, and SNP were
harvested by 0.25% trypsin/1 mM ethylenediamine tetraacetic
acid (EDTA; Nacalai Tesque, Inc.) treatment. After washingwith blocking buffer [0.1% bovine serum albumin (BSA;
Nacalai Tesque, Inc.) in phosphate‑buffered saline (PBS)],
cells were treated with H77Bf, or blocking buffer (control) for
30 min at 4˚C. Then, cells were incubated in FITC‑conjugatedanti‑dog IgG (cat. no. A18764; 1:1,000; Thermo Fisher
Scientific, Inc.) for 30 min at 4˚C. Fluorescence data werecollected by the Cell Analyzer EC800 and analyzed by EC800
software ver. 1.3.6 (Sony Corp.).
Determination of binding affinity. CHO/dHER2 and SNP weresuspended in serially diluted H77Bf (0.006‑25 µg/ml) followed
by FITC‑conjugated anti‑dog IgG (1:200). Fluorescence data

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Figure 1. Flow cytometry using H77Bf. (A) Production of H77Bf (core‑fucose‑deficient dog IgGB) from H2Mab‑77 (mouse IgG1). (B) CHO‑K1 and CHO/dHER2cells were treated with H77Bf or buffer control, followed by FITC‑conjugated anti‑dog IgG. (C) Determination of the binding affinity of H77Bf using flow
cytometry for CHO/dHER2 cells. CHO/dHER2 cells were suspended in serially diluted H77Bf, followed by the addition of FITC‑conjugated anti‑dog IgG.
Fluorescence data were analyzed using the EC800 Cell Analyzer. (D) Immunocytochemistry using H77Bf. CHO‑K1 and CHO/dHER2 cells were incubatedwith buffer control or 10 µg/ml H77Bf for 1 h, followed by the incubation with Alexa Fluor 488‑conjugated anti‑dog IgG and DAPI for 45 min. Fluorescentimages were acquired using a fluorescent microscope BZ‑X800. Scale bars, 20 µm.

were collected using the Cell Analyzer EC800. The dissocia‑
tion constant (KD) was calculated by fitting binding isotherms
to built‑in one‑site binding models in GraphPad Prism 8(GraphPad Software, Inc.).
Immunocytochemistry. Cells were fixed with 4% para‑
formaldehyde‑PBS for 10 min and quenched with 50 mM
NH4Cl in PBS with 0.2 mM Ca 2+ and 2 mM Mg2+. The cells
were blocked with blocking buffer (PBS containing 0.2 mM
Ca 2+, 2 mM Mg2+ and 0.5% BSA) for 30 min and incubatedwith 10 µg/ml of H77Bf or blocking buffer for 1 h. The

cells were further incubated with Alexa Fluor 488‑conju‑
gated anti‑dog IgG (1:400; Jackson ImmunoResearchLaboratories, Inc.) and 0.3 µM of 4',6‑diamidino‑2‑phenyl‑
indole (DAPI; Thermo Fisher Scientific, Inc.) for 45 min.
The whole processes were performed at room temperature.
Fluorescence images were acquired with a 40x objective
on a BZ‑X800 digital fluorescence microscope (Keyence
Corporation).
ADCC of H77Bf. Canine mononuclear cells (MNCs) obtained
from Yamaguchi University were resuspended in DMEM4

SUZUKI et al:  DEFUCOSYLATED MOUSE‑DOG CHIMERIC ANTI‑HER2 mAb

and 100 µg/ml of control dog IgG or H77Bf. Following incu‑
bation for 4.5 h at 37˚C, Calcein release into the medium wasmeasured.
Antitumor activity of H77Bf in xenografts of CHO‑K1,
CHO/dHER2 and SNP cells. BALB/c nude mice (female,
5 weeks old, weighing 14‑17 g) were purchased from Charles
River Laboratories, Inc. CHO‑K1, CHO/dHER2, or SNP cells(5x106 cells) were resuspended in DMEM and mixed with BD
Matrigel Matrix Growth Factor Reduced (BD Biosciences)were subcutaneously injected into the left flank of mice.
On day 8 post‑inoculation, 100 µg of H77Bf (n=8) orcontrol dog IgG (n=8) in 100 µl PBS were intraperitoneallyinjected. On days 14 and 21, additional antibody inoculations
were performed. Furthermore, on days 8, 14 and 21, canine
MNCs were injected surrounding the tumors. The tumor
volume was measured on days 7, 10, 14, 17, 21, 24 and 28 after
the injection of cells. Tumor volumes were determined aspreviously described (31,37,39‑41,50,54).
Statistical analyses. All data are expressed as mean ± stan‑
dard error of the mean (SEM). Statistical analysis was
conducted with Welch's t test for ADCC, CDC, and tumor
weight. ANOVA with Sidak's post hoc test were conducted
for tumor volume and mouse weight. All calculations were
performed using GraphPad Prism 8 (GraphPad Software,Inc.). P<0.05 was considered to indicate a statistically signifi‑
cant difference.
Figure 2. Evaluation of ADCC and CDC elicited by H77Bf. (A) ADCC
elicited by H77Bf and control dog IgG targeting CHO/dHER2 and CHO‑K1
cells. (B) CDC elicited by H77Bf and control dog IgG targeting CHO/dHER2
and CHO‑K1 cells Values are presented as the mean ± SEM. (*P<0.05;
Welch's t- test). ADCC, antibody‑dependent cellular cytotoxicity; CDC,
complement‑dependent cytotoxicity; n.s., not significant.

(Nacalai Tesque, Inc.) with 10% FBS and were used as effector
cells (37,38,42). Target cells (CHO‑K1, CHO/dHER2, andSNP) were labeled with 10 µg/ml Calcein AM (Thermo Fisher
Scientific, Inc.) (31,39‑41,43‑53). The target cells (2x104 cells)
were plated in 96‑well plates and mixed with effector canine
MNCs (effector/target cells ratio, 50), 100 µg/ml of H77Bf orcontrol dog IgG. Following incubation for 4.5 h at 37˚C, theCalcein release into the medium was analyzed using a micro‑
plate reader (Power Scan HT; BioTek Instruments, Inc.,) withan excitation wavelength (485 nm) and an emission wavelength(538 nm).
Cytolyticity (% lysis) was calculated as follows: %lysis=(E‑S)/(M‑S) x100, where ‘E’ is the fluorescence in
cultures of both effector and target cells, ‘S’ is the spontaneous
fluorescence of only target cells, and ‘M’ is the maximum fluo‑
rescence following the treatment with a lysis buffer (10 mM
Tris‑HCl (pH 7.4), 10 mM of EDTA, and 0.5% Triton X‑100).
CDC of H77Bf. Target cells (CHO‑K1, CHO/dHER2, andSNP) were labeled with 10 µg/ml Calcein AM (31,39‑41,
43‑53). The target cells (2x104 cells) were plated in 96‑wellplates and mixed with rabbit complement (final dilution 1:10;
Low‑Tox‑M Rabbit Complement; Cedarlane Laboratories,)

Results
Flow cytometric analysis against CHO/dHER2 cells using
H77Bf. In our previous study, an anti‑HER2 mAb (H2Mab‑77)
was established using cancer‑specific mAb (CasMab)method (29). H 2Mab‑77 was revealed to be very useful forflow cytometry, western blotting and immunohistochemistry(IHC) (29). In the present study, a defucosylated mouse‑dog
chimeric anti‑HER2 mAb (H77Bf ) was produced by
combining V H and V L of H2Mab‑77 with CH and CL of dog
IgG, respectively (Fig. 1A). H77Bf detected CHO/dHER2cells dose‑dependently, not parental CHO‑K1 cells (Fig. 1B),
indicating that H77Bf cross‑reacted with dHER2.
A kinetic analysis of the interactions of H77Bf with
CHO/dHER2 cells was performed via flow cytometry. As
revealed in Fig. 1C, the KD for the interaction of H77Bf with
CHO/dHER2 cells was 7.5x10 ‑10 M, suggesting that H77Bf
exhibits high affinity for CHO/dHER2 cells.
Immunocytochemical analysis against CHO/dHER2 cells
using H77Bf. It was examined whether H77Bf is applicablefor immunocytochemistry. The H77Bf specificity was evalu‑
ated by using CHO/dHER2 and CHO‑K1 cells. As revealedin Fig. 1D, H77Bf detected dHER2 on CHO/dHER2 cells,
but not CHO‑K1 cells. Buffer control showed no signal
on both CHO/dHER2 and CHO‑K1 cells. These results
suggested that H77Bf recognizes exogenous dHER2 in
immunocytochemistry.
H77Bf‑mediated ADCC and CDC in CHO/dHER2 cells. It
was investigated whether H77Bf was capable of mediating

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Figure 3. Antitumor activity of H77Bf. (A and B) Evaluation of tumor volume in (A) CHO/dHER2 and (B) CHO‑K1 xenograft models. CHO/dHER2 andCHO‑K1 cells (5x106 cells) were subcutaneously injected into mice. On day 8, 100 µg of H77Bf or control dog IgG were injected intraperitoneally intomice. Additional antibodies were injected on days 14 and 21. Mononuclear cells were also injected surrounding the tumors on days 8, 14 and 21. The tumor
volume was measured on days 7, 10, 14, 17, 21, 24 and 28 after the injection. Values are presented as the mean ± SEM. **P<0.01 (ANOVA and Sidak's multiple
comparisons test). (C and D) Tumor weight (day 28) was measured from excised xenografts of (C) CHO/dHER2 and (D) CHO‑K1. Values are presented as themean ± SEM. *P<0.05 (Welch's t-test). (E and F) Appearance of resected tumors of (E) CHO/dHER2 and (F) CHO‑K1 xenografts from the control dog IgG
and H77Bf treated groups on day 28 (scale bar, 1 cm). n.s., not significant.

ADCC against CHO/dHER2 cells. H77Bf showed ADCC
(31.8% cytotoxicity) against CHO/dHER2 cells more effec‑
tively than the control dog IgG (13.2% cytotoxicity; P<0.05).
There was no difference between H77Bf and control dog IgG
about ADCC against CHO‑K1 (Fig. 2A).
It was then examined whether H77Bf could exert CDC
against CHO/dHER2 cells. As revealed in Fig. 2B, H77Bf
elicited a higher degree of CDC (50.7% cytotoxicity) in
CHO/dHER2 cells compared with that elicited by control
dog IgG (33.1% cytotoxicity; P<0.05). There was no differ‑ence between H77Bf and control dog IgG about CDC
against CHO‑K1 (Fig. 2B). These results demonstrated that

H77Bf exhibited higher levels of ADCC and CDC against
CHO/dHER2 cells.
Antitumor effects of H77Bf in the mouse xenografts of
CHO/dHER2 cells. In the CHO/dHER2 xenograft tumor,
H77Bf and control dog IgG were intraperitoneally injected
into mice on days 8, 14 and 21, following the CHO/dHER2cells injection. On days 7, 10, 14, 17, 21, 24 and 28 after the
injection, the tumor volume was measured. The H77Bf
administration resulted in a significant reduction of tumors ondays 24 (P<0.01) and 28 (P<0.01) compared with that of thecontrol dog IgG (Fig. 3A). The H77Bf administration resulted

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Figure 4. Body weights and appearance of the mice. (A and B) Body weights of mice implanted with (A) CHO/dHER2 and (B) CHO‑K1 xenografts on days 7,
10, 14, 17, 21, 24 and 28 (ANOVA and Sidak's multiple comparisons test). (C and D) Body appearance of (C) CHO/dHER2 and (D) CHO‑K1‑implanted miceon day 28 (scale bar, 1 cm). n.s., not significant.

in a 65% reduction of the volume compared with that of thecontrol dog IgG on day 28 post‑injection.
The weight of CHO/dHER2 tumors treated with H77Bf
was significantly lower than that treated with control dog
IgG (71% reduction; P<0.05; Fig. 3C). CHO/dHER2 tumorsthat were resected from mice on day 28 are demonstrated inFig. 3E.
In the CHO‑K1 xenograft models, H77Bf and control
dog IgG were injected intraperitoneally into mice on days 8,
14 and 21 after the injection of CHO‑K1 cells. The tumor
volume was measured on days 7, 10, 14, 17, 21, 24 and 28
after the injection of cells. No difference was observed
between H77Bf and control dog IgG about CHO‑K1 tumor
volume (Fig. 3B) and CHO‑K1 tumor weight (Fig. 3D).
CHO‑K1 tumors that were resected from mice on day 28 aredemonstrated in Fig. 3F.
The body weights loss and skin disorder were notobserved in CHO/dHER2 (Fig. 4A) and CHO‑K1 (Fig. 4B)
tumor‑bearing mice. The mice on day 28 about CHO/dHER2
and CHO‑K1 were shown in Fig. 4C and D, respectively.
Flow cytometry and immunocytochemical analysis against
SNP cells using H77Bf. As demonstrated in Fig. 5A, H77Bf
detected SNP cells dose‑dependently. A kinetic analysis of

the binding of H77Bf to SNP cells was performed via flow
cytometry. The KD for the interaction of H77Bf with SNP cells
was 7.2x10‑10 M (Fig. 5B), suggesting that H77Bf shows highaffinity for SNP cells.
Immunocytochemical analysis was then performed using
H77Bf for SNP cells. As a result, H77Bf detected dHER2 on
SNP cells (Fig. 5C). Buffer control detected no signal on SNP
cells. These results indicated that H77Bf recognizes endog‑
enous dHER2 in immunocytochemistry.
H77Bf‑mediated ADCC and CDC in SNP cells. It was investi‑
gated whether H77Bf was capable of mediating ADCC against
SNP cells. As revealed in Fig. 5D, H77Bf showed ADCC
(24.8% cytotoxicity) against SNP cells more potently than did
the control dog IgG (6.3% cytotoxicity; P<0.05). It was next
investigated whether H77Bf exhibited CDC against SNP cells.
H77Bf induced a higher degree of CDC (63.9% cytotoxicity)
in SNP cells compared with that induced by control dog IgG(45.7% cytotoxicity; P<0.05) (Fig. 5D). These results demon‑
strated that H77Bf exhibited higher levels of ADCC and CDC
against SNP cells.
Antitumor effects of H77Bf on SNP xenografts. In the SNP
xenograft models, H77Bf and control dog IgG were injected

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Figure 5. Flow cytometry, ADCC and CDC activity of H77Bf against canine mammary gland tumor cell line, SNP cells. (A) SNP cells were treated with
H77Bf or buffer control, followed by FITC‑conjugated anti‑dog IgG. (B) Determination of the binding affinity of H77Bf for SNP cells using flow cytometry.
SNP cells were suspended in 100 µl of serially diluted H77Bf, followed by the addition of FITC‑conjugated anti‑dog IgG. Fluorescence data were collectedusing the EC800 Cell Analyzer. (C) Immunocytochemistry using H77Bf. SNP cells were incubated with buffer control or 10 µg/ml H77Bf for 1 h, followedby the incubation with Alexa Fluor 488‑conjugated anti‑dog IgG and DAPI for 45 min. Fluorescent images were acquired using a fluorescent microscope
BZ‑X800 (scale bars, 20 µm). (D) ADCC and CDC elicited by H77Bf and control dog IgG targeting SNP cells. Values are presented as the mean ± SEM.
*
P<0.05; Welch's t-test). ADCC, antibody‑dependent cellular cytotoxicity; CDC, complement‑dependent cytotoxicity.

intraperitoneally on days 8, 14 and 21, after the injection of
SNP cells. The tumor volume was measured on days 7, 10, 14,
17, 21, 24 and 28 after the injection. The H77Bf administration
resulted in a significant reduction in tumor growth on days 10(P<0.01), 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(Fig. 6A). The H77Bf administration resulted in a 47% reduc‑tion of tumor volume compared with that of the control dog
IgG on day 28.
Tumors from the H77Bf‑treated mice weighed significantly
less than those from the control dog IgG‑treated mice (35%
reduction; P<0.05, Fig. 6B). Tumors that were resected frommice on day 28 are demonstrated in Fig. 6C.
The body weights loss and skin disorder were not observedin SNP tumor‑bearing mice (Fig. 7A). The mice on day 28
about SNP xenograft were demonstrated in Fig. 7B.

Discussion
Human mAbs that exhibit cross‑reactivity to dog have been
investigated. It has been suggested that cetuximab (anti‑EGFR)
and trastuzumab (anti‑HER2) can bind to certain canine
cancer cell lines (55). The clinical relevance though is limited
considering that those antibodies, such as trastuzumab, mostly
work through ADCC (56). Furthermore, there is a problem
that the humanized mAbs will induce an anti‑human immune
response in dogs. Therefore, the caninization of mAbs (onlythe complementarity determining regions are non‑canine) is
essential to develop antibody therapy for dog. Some caninizedmAbs have received conditional approval by the UnitedStates Department of Agriculture for lymphoma (for example
Blontress, targeting CD20; and Tactress, targeting CD52).
However, no peer‑reviewed clinical evidence of efficacy for the

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SUZUKI et al:  DEFUCOSYLATED MOUSE‑DOG CHIMERIC ANTI‑HER2 mAb

Figure 7. Body weights and appearance of the mice. (A) Body weights ofmice implanted with SNP xenografts on days 7, 10, 14, 17, 21, 24 and 28(ANOVA and Sidak's multiple comparisons test). (B) Body appearance of
SNP‑implanted mice on day 28 (scale bar, 1 cm). n.s., not significant.
Figure 6. Antitumor activity of H77Bf against SNP xenograft. (A) Evaluationof tumor volume in SNP xenograft models. SNP cells (5x10 6 cells) were
injected subcutaneously into mice. On day 8, 100 µg of H77Bf or control
dog IgG in 100 µl PBS were injected intraperitoneally into mice. Additional
antibodies were injected on days 14 and 21. Mononuclear cells were also
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 inoculation. Values
are presented as the mean ± SEM. **P<0.01 (ANOVA and Sidak's multiple
comparisons test). (B) Tumor weight (day 28) was measured from excised
SNP xenografts. Values are presented as the mean ± SEM. *P<0.05 (Welch's
t-test). (C) Appearance of resected SNP xenografts from the control dog IgG
and H77Bf treated groups on day 28 (scale bar, 1 cm).

mAb has been published (57). In the present study, caninizedmAb, H77Bf was developed from anti‑HER2 mAb H2Mab‑77.
Among IgG subclasses (A, B, C and D) in dogs, the B and
D subclasses were reported to be involved in ADCC (58).
Therefore, B type dog IgG was converted and a defucosylated
mAb was produced, which has been shown to exhibit morepotent ADCC activity through binding to FcγRIIIa on NK
cells (59). The cross‑reactivity and binding affinity of H77Bf
to CHO/dHER2 and SNP cells were first confirmed, and it was

found that H77Bf possesses comparable high binding affinity
to CHO/dHER2 (7.5x10 ‑10 M) and SNP (7.2x10 ‑10 M) cells,
compared with human cancer A431 (2.1x10 ‑9 M by H2Mab‑77)
and SK‑BR‑3 (7.3x10 ‑9 M by H2Mab‑77) cells, as previously
reported (29). The quantitative analysis is considered to be
essential to apply a human antibody to dog.
In vivo administration of H77Bf and canine MNC resulted
in significant growth inhibition for CHO/dHER2 and SNP cells.
These results provided evidences to support the suitability of
H77Bf as a promising antibody therapy against canine cancers.
The ADCC activity was also confirmed in vitro using canine
MNCs, suggesting that ADCC activity could contribute to theantitumor activity of H77Bf. ADCC in humans is executed
predominantly by NK cells through the FcγR that binds to the
IgG1 or IgG3 subclass (60). The FcγR‑like receptors have notbeen described on canine NK cells. Recently, a cell line‑based
assay to measure the ADCC of a canine therapeutic antibody
was reported (61). The aforementioned study established ahuman NK cell line, NK‑92 cells expressed with canine FcγR
which can be used as effector cells. This system will contribute

ONCOLOGY REPORTS 48: 154, 2022

to the understanding of NK cell‑mediated target cell lysis bycanine therapeutic antibodies. Since the knowledge about
canine NK cells is incomplete, further studies are needed
to reveal the contribution of NK cells to ADCC in dogs.
Furthermore, direct cytotoxic mechanisms by the complement
system in dogs is also to be determined.
Drug‑conjugated mAbs rely on direct cytotoxicityof the payloads through endocytosis of receptor‑boundmAbs‑drug conjugate (62,63). Trastuzumab deruxtecan(T‑DXd, DS‑8201) is a HER2‑targeting antibody conju‑
gated with a novel DNA topoisomerase I inhibitor (64).
T‑DXd showed promising clinical outcomes in patientswith metastatic breast cancer, who had received multiple
anti‑HER2‑targeting regimens (65). Currently, the clinicalefficacy and safety of T‑DXd have been evaluated in various
clinical trials. T‑DXd have been approved in not only
HER2‑positive breast cancer (65‑67), but also HER2‑mutant
lung cancer (66). A mouse‑canine chimeric mAb against
dog podoplanin (68‑70) (P38B) conjugated with emtansine
as the payload (P38B‑DM1) was previously generated andchallenged for tumor therapy. P38B‑DM1 showed cytotox‑
icity to podoplanin‑expressing cells and exhibited higher
antitumor activity than P38B in the xenograft model (71).
Therefore, H77B‑drug conjugate is one more option to treatdHER2‑positive CMT. Recently, FDA‑approved humanimmune checkpoint inhibitor against PD‑1 and PD‑L1 areused in canine tumor treatment (72‑74); the combination of
immune checkpoint inhibitors with other antibody‑drugs is
expected to be more effective. H77Bf could contribute to
the development of canine cancer treatment, which can be
feedback for human cancer treatment.
IHC has played a critical role as a diagnostic tool for theidentification of neoplasms with conventional histopathology.
In human breast cancer pathology, IHC is routinely used toassist with the prognosis and to determine the specific treat‑ment (e.g. trastuzumab) for patients. Although IHC is notroutinely used in CMTs, an increasing number of studies
have been looking for reliable diagnostic and/or prognostic
IHC biomarkers including dHER2 (21). A positive correla‑tion between dHER2 in serum and tissue expression (byIHC) was reported (26). There is also a positive correlation
between dHER2 expression and tumor mitotic index, highhistological grade and size (75). However, not all studies haveconfirmed this, and no difference between dHER2 expression
in non‑neoplastic and neoplastic lesions was observed (76).
Furthermore, in contrast to HER2‑positive breast cancer inhuman, dHER2 amplification and HER2‑enrichment subtype
are not observed through whole‑exome and transcriptome
analyses of 191 spontaneous CMTs (77). Therefore, thestandardization of dHER2 IHC is essential since those IHC
analyses were performed by different Abs. Our established
H2Mab‑77 mAb is available for IHC (29), and its caninizedmAb H77Bf exerts the antitumor activity against dHER2
positive cells, which could contribute to both diagnosis andtherapy for dHER2‑positive canine tumors.
Acknowledgements
The authors would like to thank Ms. Miyuki Yanaka, Mr.
Takuro Nakamura, Mr. Yu Komatsu and Ms. Saori Handa

9(Department of Antibody Drug Development, Tohoku
University Graduate School of Medicine) for technicalassistance of in vitro experiments, and Mr. Shun‑ichi Ohba
and Ms. Akiko Harakawa [Institute of Microbial Chemistry(BIKAKEN), Numazu, Microbial Chemistry Research
Foundation] for technical assistance of animal experiments.
Funding
The present study was supported in part by Japan Agency
for Medical Research and Development (AMED; grantnos: JP22ama121008, JP21am0401013, JP22bm1004001,
JP22ck0106730 and JP21am0101078).
Availability of data and materials
All data generated or analyzed during this study are includedin this published article.
Authors' contributions
TO, TT, MS and TA performed the experiments. MKK, MK
and YK designed the experiments. TM prepared canine
MNCs. TA, HS, TY and YK analyzed the data. HS and YK
wrote the manuscript. All authors read and approved thefinal manuscript and agree to be accountable for all aspectsof the research in ensuring that the accuracy or integrityof any part of the work are appropriately investigated andresolved.
Ethics approval and consent to participate
The animal study protocol was approved (approval
no. 2021‑056) by the Institutional Committee for Experimentsof the Institute of Microbial Chemistry (Numazu, Japan).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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