MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 4, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0025

An Anti-HER2 Monoclonal Antibody H2Mab-41 Exerts
Antitumor Activities in Mouse Xenograft Model
Using Dog HER2-Overexpressed Cells

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Nami Tateyama,1,* Teizo Asano,1,* Tomokazu Ohishi,2,* Junko Takei,1 Hideki Hosono,1 Ren Nanamiya,1Tomohiro Tanaka,1 Masato Sano,1 Masaki Saito,3 Manabu Kawada,2 Mika K. Kaneko,1 and Yukinari Kato1,3,4,iOverexpression of human epidermal growth factor receptor 2 (HER2) has been reported in a variety of cancer
types, including breast, lung, gastric, pancreatic, and colorectal cancers. Trastuzumab, a humanized anti-HER2
monoclonal antibody (mAb), has been shown to provide significant survival benefits in HER2-overexpressing
breast cancer and gastric cancer patients. Previously, an anti-HER2 mAb, H2Mab-41 (IgG2b, kappa), wasdeveloped in our laboratory and its antitumor activity was demonstrated in mouse xenograft models of humancolon cancer. The present study aimed to investigate the ability of H2Mab-41 to induce antibody-dependent
cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dog HER2 (dHER2)-overexpressed cell lines, and thus exert its antitumor activity against dHER2-overexpressed tumors in vivo. Flowcytometry results demonstrated the cross-reactivity of H2Mab-41 with dHER2. Further evaluation of interaction
between H2Mab-41 and dHER2-overexpressed CHO-K1 (CHO/dHER2) cells indicated moderate binding affinity of H2Mab-41 toward dHER2, with a dissociation constant (KD) of 2.6 · 10-8 M. In vitro analysis revealed
that the administration of H2Mab-41 induced high levels of ADCC and CDC in CHO/dHER2 cells. Furthermore, intraperitoneal administration of H2Mab-41 in mouse xenograft models of CHO/dHER2 resulted insignificant inhibition of tumor development compared to the control mouse IgG. Thus, the findings of thepresent study demonstrated the in vivo safety and efficacy of H2Mab-41, highlighting its suitability to be
included as a part of a therapeutic regimen for dHER2-expressing canine cancers.
Keywords: HER2, monoclonal antibody, ADCC, CDC, antitumor activity

IntroductionHuman epidermal growth factor receptor 2(HER2) is a type I transmembrane glycoprotein that isoverexpressed in several cancer types, especially breast
cancer.(1) HER2 belongs to epidermal growth factor receptor(EGFR) family, which comprises of four main members,
namely EGFR (HER1/ErbB1), HER2 (ErbB2/neu), HER3
(ErbB3), and HER4 (ErbB4).(2) The EGFR family members
transduce extracellular signals into intracellular signaling
pathways via activation of tyrosine kinase domain. These
receptor tyrosine kinases play a critical role in the regulationof cell proliferation, differentiation, survival, and migration.(3) The binding of ligand to the extracellular domain of

the receptors promotes the formation of homo- or heterodimers between the EGFR family receptors.(4) Interestingly,
HER2 is devoid of any ligand-binding extracellular domain,and it is represented as an activated structure in the absence ofligand binding.(5–7) Generally, HER2 is expressed in a varietyof cell types, excluding the cells of hematopoietic origin.(8)The overexpression of HER2 has been reported in a variety ofcancer types, including breast,(9,10) gastric,(11) pancreatic,(12)lung,(13) and colorectal cancers.(14) In HER2-positive breast
cancer patients, the overexpression of this oncogene was foundto be associated with poor prognosis and aggressive tumor
phenotype.(9,10) Trastuzumab and pertuzumab are humanizedanti-HER2 monoclonal antibodies (mAbs) that are widelyused in the treatment of HER2-positive breast cancer.(15–17)

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Japan.
3
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
4
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
*These authors contributed equally to this work.
i
ORCID ID (https://orcid.org/0000-0001-5385-8201).
2

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ANTITUMOR ACTIVITIES OF ANTI-HER2 MAB

Cancer is the leading cause of death in dogs. Annual incidence rate of cancer in dogs is estimated to be *30%,
which is similar to humans.(18,19) In fact, overexpression of
HER2 has also been reported in a variety of canine tumors,
including primary lung cancer,(20) osteosarcoma,(21) mammary carcinoma,(22) bladder transitional cell carcinoma,(23)and anal sac gland carcinoma.(24) The cancer therapy for
veterinary oncology treatment coevolved with human cancer
treatment. In the past few decades, various HER2-targetingtherapeutic agents, such as Lapatinib and recombinant Listeria vaccines, have been developed and utilized for cancer
treatment. Lapatinib is a low-molecular-weight tyrosine kinase inhibitor that targets EGFR and HER2. These HER2targeted therapies have been shown to exert an antitumor
effect on canine HER2 (dHER2)-positive cancers.(25,26)
Thus, HER2 could be used as a potential therapeutic targetand diagnostic marker for canine cancers. Previously,
H2Mab-41, an anti-HER2 mAb developed in our laboratory
was found to exert antitumor activity in a mouse xenograftmodel for human colon cancer.(27) The present study aimed
to investigate the ability of H2Mab-41 to induce antibodydependent cellular cytotoxicity (ADCC), complementdependent cytotoxicity (CDC), and antitumor efficacy indHER2-overexpressed cells.
Materials and Methods
Antibodies

Anti-HER2 mAb H2Mab-41 (mouse IgG2b, kappa) was
developed according to the previously established procedure.(27) The resulting antibody was purified using Protein GSepharose (GE Healthcare Bio-Sciences, Pittsburgh, PA).
Mouse IgG (Cat. No. I8765) and IgG2b (Cat. No. M1395)
were purchased from Sigma-Aldrich (St. Louis, MO).
Cell lines

CHO-K1 cells were obtained from the American Type
Culture Collection (Manassas, VA). Dog HER2 (dHER2;
Accession No. NM_001003217)-overexpressed CHO-K1
cell line (CHO/dHER2) was established via transfection ofpCAG/3xRIEDL-dHER2 into CHO-K1 cells using Neontransfection system (Thermo Fisher Scientific, Inc., Waltham, MA). Here, 3xRIEDL sequence represented 15 aminoacid sequence (RIEDLRIEDLRIEDL).(28) RIEDL tag is a
novel affinity tag that is used for the detection and one-steppurification of membrane proteins. For cell culture, CHO-K1
and CHO/dHER2 were cultured in Roswell Park Memorial
Institute (RPMI)-1640 medium (Nacalai Tesque, Inc., Kyoto,
Japan), supplemented with 10% heat-inactivated fetal bovine
serum (FBS; Thermo Fisher Scientific, Inc.), 100 U/mL of
penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.). The cell lines were maintained at 37C in a humidified atmosphere under 5% CO2.
Animals

To ensure minimal animal suffering and distress in thelaboratory, all animal experiments were performed according
to the established guidelines and regulations. Animal experiments for ADCC and antitumor activity were approved by
the Institutional Committee for Experiments of the Instituteof Microbial Chemistry (Permit No. 2021-019 and 2021-007

185

for ADCC assays and antitumor experiments, respectively).
Mice were maintained under a specific pathogen-free environment, at a temperature of 23C – 2C with 55% – 5%
relative humidity and 11/13 hours light/dark cycle. The animals had free access to food and water during the entire
course of the study. Mice were regularly monitored for healthand weight every 2 or 5 days during the 3-week period foreach experiment. Loss of >25% of original body weight
and/or a maximum tumor size >3000 mm3 were identified as
humane endpoints for euthanasia. Mice were euthanized bycervical dislocation, and death was verified by respiratory
and cardiac arrest.
Flow cytometry

For cell harvesting, CHO-K1 and CHO/dHER2 cells weretreated with a solution of 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.) for a brief
duration (1–2 minutes). Posttrypsinization, the cells were
washed with blocking buffer, comprising 0.1% bovine serum
albumin in phosphate-buffered saline (PBS). Further, thecells were treated with 1 lg/mL of H2Mab-41 or control
blocking buffer for 30 minutes at 4C. This was followed byaddition of Alexa Fluor 488-conjugated anti-mouse IgG at adilution of 1:1000 (Cat. No. 4408S; Cell Signaling Technology, Inc., Danvers, MA, USA). The cells were incubatedfor 30 minutes at 4C. Fluorescence data were acquired using
EC800 Cell Analyzer (Sony Corp., Tokyo, Japan).
Determination of the binding affinity

To evaluate the binding affinity, CHO/dHER2 cells werefirst suspended in 100 lL of serially diluted H2Mab-41(0.006–100 lg/mL). This was followed by the addition of
Alexa Fluor 488-conjugated anti-mouse IgG (1:200 dilution;
Cell Signaling Technology, Inc.). Fluorescence data wereacquired using EC800 Cell Analyzer. The dissociation constant (KD) was calculated by fitting the binding isotherms
using the built-in one-site binding models in GraphPad Prism
8 (GraphPad Software, Inc., La Jolla, CA).
Antibody-dependent cellular cytotoxicity

For ADCC assay, 5-week-old female BALB/c (n = 5) nude
mice (weighing 14–17 g) were purchased from Charles River
Laboratories, Inc., (Kanagawa, Japan). Spleen cells wereisolated from these mice and used as the source of mononuclear cells to evaluate ADCC, as previously described.(29)Mice were euthanized by cervical dislocation, and the spleens
were removed aseptically. To obtain single-cell suspensions,the spleen tissues were forcefully passed through a sterile cellstrainer (352360, BD Falcon; Corning, New York, NY) usinga syringe. Erythrocytes were exposed to ice-cold distilledwater for 10 seconds to ensure complete lysis. The resulting
splenocytes were washed with RPMI-1640 and resuspendedin RPMI-1640 supplemented with 10% FBS. This cell
preparation was further used as effector cells. CHO/dHER2cells were labeled with 10 mg/mL Calcein AM (Thermo
Fisher Scientific, Inc.) and resuspended in the same medium.
CHO/dHER2 cells were seeded in 96-well plates at a densityof 2 · 104 cells/well. These cells were mixed with effector
cells (effector/target cells ratio, 100) and 100 mg/mL of
H2Mab-41 or control mouse IgG2b, and incubated at 37C for186

5 hours. Posttreatment, the release of Calcein into the supernatant was measured in terms of fluorescence intensity ineach well, measured using a microplate reader (Power Scan
HT; BioTek Instruments, Inc., Winooski, VT) with excitationand emission wavelengths of 485 and 538 nm, respectively.
Cytolytic activity (%lysis) was calculated using the equation%lysis = (E - S)/(M - S) · 100, where ‘‘E’’ is the fluorescenceintensity for the combined cultures of target and effector
cells, ‘‘S’’ represents spontaneous fluorescence of target cells
alone, and ‘‘M’’ denotes maximum fluorescence measured in
all cells postlysis. The cells were lysed using a buffer comprising of 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4), and10 mM of EDTA.

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Complement-dependent cytotoxicity

For CDC evaluation, CHO/dHER2 cells were seeded in 96well plates at a density of 2 · 104 cells/well. These cells weremixed with rabbit complement reagent (final dilution 1:10;
Low-Tox-M Rabbit Complement; Cedarlane Laboratories,

TATEYAMA ET AL.

Hornby, Ontario, Canada) and 100 lg/mL of H2Mab-41 orcontrol mouse IgG2b. The cells were incubated at 37C for 5
hours, and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;
innersalt] assay was performed using a CellTiter 96 AQueous One
Solution Cell Proliferation Assay Kit (Promega, Madison, WI).
Antitumor activity of H2Mab-41 in mice xenografts
for CHO/dHER2 cells

For antitumor activity, 16 female BALB/c nude mice, aged
5 weeks and weighing 14–17 g, were purchased from Charles
River Laboratories, Inc. The animals were housed in the institutional animal facility and experiments were conducted at
the age of 6 weeks. For tumor inoculation, 0.3 mL of
CHO/dHER2 cells (1.33 · 108 cells/mL) in RPMI-1640 was
mixed with 0.5 mL of BD Matrigel Matrix Growth Factor
Reduced (BD Biosciences, San Jose, CA), and 100 lL of theresulting suspension (5 · 106 cells) was injected subcutaneously into the left flanks of the mice. On day 6 postinoculation,

FIG. 1. Flow cytometry analysis for interaction between H2Mab-41 and CHO/dHER2 cells. (A) CHO/dHER2 and CHO-K1
cells were treated with H2Mab-41 or blocking buffer control, followed by addition of secondary antibodies. (B) Flow cytometry analysis to determine binding affinity of H2Mab-41 for CHO/dHER2 cells. CHO/dHER2 cells were suspended in 100 mLof serially diluted H2Mab-41, followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG. For both experiments,
fluorescence data were acquired using EC800 Cell Analyzer. HER2, human epidermal growth factor receptor 2.

ANTITUMOR ACTIVITIES OF ANTI-HER2 MAB

187

mice received intraperitoneal injections of 100 lg of H2Mab41 (n = 8), or control mouse IgG (n = 8) in 100 mL PBS. Additional antibody treatments were performed on days 14 and
21. At day 25 after tumor inoculation, all mice were euthanized by cervical dislocation, and tumor diameters and volumes were determined as described previously.(29)
Statistical analyses

All data are expressed as mean – standard error of themean. Statistical analyses for ADCC and CDC experiments
were conducted using Welch’s t-test. ANOVA and Sidak’s
multiple comparisons tests were performed for tumor volume
and mouse weight. For tumor weight, Welch’s t-test was
conducted. All calculations were performed using GraphPad
Prism 7 (GraphPad Software, Inc.). A p-Value of < 0.05 wasconsidered to be statistically significant.

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Results
Characterization of H2Mab-41 for CHO/dHER2

The cross-reactivity of H2Mab-41 for CHO/dHER2 wasanalyzed using flow cytometry. As shown in Figure 1A,
H2Mab-41 successfully detected CHO/dHER2, however,parental CHO-K1 cells remained undetected.
A kinetic analysis for the interaction of H2Mab-41 with
CHO/dHER2 cells was performed using flow cytometry. As
shown in Figure 1B, the interaction between H2Mab-41 andCHO/dHER2 cells was characterized by a KD of 2.6 · 10-8
M. Thus, H2Mab-41 was found to have moderate affinity
toward CHO/dHER2 cells.
H2Mab-41-mediated ADCC and CDC
in CHO/dHER2 cells

The present study investigated the ability of H2Mab-41 tomediate ADCC in CHO/dHER2 cell line. As shown in
Figure 2A, H2Mab-41 was found to exert higher ADCC activity (34% cytotoxicity; p < 0.05) against CHO/dHER2 cells
compared to the control mouse IgG2b (7.2% cytotoxicity).
Further, the ability of H2Mab-41 to mediate CDC against
CHO/dHER2 cells was also evaluated. As shown in
Figure 2B, H2Mab-41 induced a higher CDC activity (52%
cytotoxicity; p < 0.05) against CHO/dHER2 cells comparedto the control mouse IgG2b (18% cytotoxicity). Thus, thetreatment of HER2-overexpressed cells with H2Mab-41 resulted in significantly higher levels of ADCC and CDC
compared to the control mouse IgG2b.
Antitumor activities of H2Mab-41 in the mouse
xenograft model for CHO/dHER2 cells

To evaluate the antitumor activity of H2Mab-41, mice
xenograft models were generated by subcutaneous implantation of CHO/dHER2 cells into left flanks of female nude
mice. Mice received intraperitoneal injections of H2Mab-41(100 lg) or control mouse IgG (100 lg) at days 6, 14, and 21
posttumor inoculation. The tumor volume was measured ondays 6, 11, 14, 18, 21, and 25 posttumor inoculation. The
administration of H2Mab-41 resulted in a significant reduction in tumor development compared to the mice receiving
control mouse IgG. The tumor reduction was particularly
significant on days 18 ( p < 0.01), 21 ( p < 0.01), and 25

FIG. 2. Evaluation of H2Mab-41 mediated ADCC and
CDC activity in CHO/dHER2 cells. (A) ADCC elicited by
H2Mab-41 or control mouse IgG2b targeting CHO/dHER2cells. (B) CDC elicited by H2Mab-41 or control mouse
IgG2b targeting CHO/dHER2 cells. Values are shown asmean – SEM. Asterisks indicate statistical significance(*p < 0.05; Welch’s t-test). ADCC, antibody-dependent
cellular cytotoxicity; CDC, complement-dependent cytotoxicity; SEM, standard error of the mean.
( p < 0.01), posttumor inoculation (Fig. 3A). After 25 days oftumor inoculation, 80% reduction in the tumor volume wasrecorded in the mice treated with H2Mab-41 compared to themice receiving control mouse IgG. Similarly, tumor weights
were also found to be significantly lower in the group treatedwith H2Mab-41 compared to the control group (84% reduction; p < 0.05, Fig. 3B). On day 25 posttumor inoculation,
mice were euthanized and tumors were resected. The imagesof the isolated tumors are shown in Figure 3C. No significant
differences were reported in the total body weights for thetwo groups (Fig. 3D). The body appearance of mice on day 25
postinoculation is shown in Figure 3E. Thus, administrationof H2Mab-41 in mice xenograft for CHO/dHER2 resulted insignificant reduction in tumor growth.
Discussion

Cancer is an evolutionary disease, which involves various
genetic alterations like gene amplification and/or mutation.(30,31) Such alterations have been shown to influenceTATEYAMA ET AL.

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FIG. 3. Evaluation of antitumor activity of H2Mab-41 in mice xenograft model for CHO/dHER2. (A) CHO/dHER2 cells(5 · 106 cells) were injected subcutaneously into the left flank. Mice received intraperitoneal injections of 100 mg of H2Mab41 or control mouse IgG in 100 mL phosphate-buffered saline on days 6, 14, and 21 posttumor inoculation. The tumor
volume was measured on days 6, 11, 14, 18, 21, and 25, posttumor inoculation. Values are shown as means – SEM.
Asterisks indicate statistical significance (**p < 0.01; ANOVA and Sidak’s multiple comparisons test). (B) On day 25
postinoculation, tumors were resected from H2Mab-41 and control mouse IgG groups, and tumors were weighed. Values areshown as mean – SEM. Asterisk indicates statistical significance (*p < 0.05, Welch’s t-test). (C) Images for tumors obtained
from CHO/dHER2 mice xenografts belonging to control mouse IgG and H2Mab-41 groups, resected on day 25. Scale bar,
1 cm. (D) Body weights of the mice implanted with CHO/dHER2 xenografts were recorded on days 6, 11, 14, 18, 21, and
25. n.s., not significant. (E) Body appearance of mice on day 25. Scale bar, 1 cm.

patient prognosis and survival.(30–32) Some of these genetic
alterations induce activation of driver mutations that result in
the constitutive upregulation of signaling pathways, conferring
advantages of cell proliferation, cell viability, and survival tothe cancerous cells.(33,34) In the past few years, driver mutations

have emerged as potential targets for molecular-targeted therapies for the management of various cancers.(35,36) Among
various driver mutations, HER2 overexpression or gene amplification has been shown to be associated with poor prognosis
in cancer patients.(37–39) Thus, the patients with HER2-driven

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ANTITUMOR ACTIVITIES OF ANTI-HER2 MAB

signaling are considered to be suitable candidates for HER2targeted therapy. In fact, administration of HER2-targeting
agents in HER2-expressing metastatic breast cancer and gastriccancer patients resulted in dramatic improvement in the diseaseprognosis.(40,41)
Trastuzumab deruxtecan (T-DXd, DS-8201) is a HER2targeting antibody-drug conjugate, comprising anti-HER2
mAb and a novel DNA topoisomerase I inhibitor.(42) Administration of T-DXd showed impressive clinical outcomesin metastatic breast cancer patients, who had received priortreatment involving multiple anti-HER2-targeting regimens.(43) These clinical outcomes encouraged the approval of
T-DXd in the United States under accelerated approval forthe treatment of patients with HER2-positive unresectable ormetastatic breast cancer, who had previously received morethan two anti-HER2-based therapies.(44) Currently, the clinical efficacy and safety of T-DXd is being evaluated in various stages of clinical trials for a variety of HER2-expressing
tumors, with the aim to expand the usage of this therapeutic
agent to other tumor types.
In canine tumors, the overexpression of HER2 has beenreported in osteosarcoma (86% of the cell lines and 40% of
the tissue samples),(21) mammary carcinoma (21% of thetissue samples),(22) bladder transitional cell carcinoma (56%of the tissue samples),(23) and anal sac gland carcinoma (80%of the tissue samples).(24) Interestingly, administration of arecombinant Listeria vaccine expressing a chimeric human
HER2 resulted in the induction of HER2-specific immunity,
reduction in the incidences of metastasis, and prolonged patient survival in a phase I trial for canine osteosarcoma.(45)
These clinical outcomes encouraged the evaluation of antidHER2 mAb as a therapeutic agent for canine cancers.
One of the most important objectives of the present study
was to demonstrate the cytotoxic activity of H2Mab-41 against
HER2-expressing cells. In vivo administration of anti-HER2
mAb resulted in significant growth inhibition for CHO/dHER2cells (Fig. 3). These results provided evidences to support thesuitability of H2Mab-41 as a promising antibody therapy
against canine cancers. Since the antitumor activity of H2Mab41 was only established against dHER2-overexpressed cells in
the present study, future studies involving various types ofdHER2-expressing canine cancers are necessary to develop
effective antibody therapies for canine cancers.
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: JP21am0401013 (to Y.K.) and JP21am0101078(to Y.K.), and by the Japan Society for the Promotion of Science ( JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Grant Nos. 21K15523 (to T.A.), 21K07168(to M.K.K.), 20K16322 (to M.Sano), and 19K07705 (to Y.K.).
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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: May 19, 2021
Accepted: July 14, 2021