MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 3, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0011

Original Articles

H2Mab-19 Anti-Human Epidermal Growth Factor Receptor 2
Monoclonal Antibody Therapy Exerts Antitumor Activity
in Pancreatic Cancer Xenograft Models

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Yukinari Kato,1,2 Tomokazu Ohishi,3 Masato Sano,1 Teizo Asano,1 Yusuke Sayama,1Junko Takei,1 Manabu Kawada,3 and Mika K. Kaneko1

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in breast cancer,
gastric, lung, colorectal, oral, and pancreatic cancers. HER2 expression is associated with poor clinical outcomes. An anti-HER2 humanized antibody, trastuzumab, has improved survival rates in patients with HER2overexpressing breast and gastric cancers. Previously, we established a novel anti-HER2 monoclonal antibody(mAb), H2Mab-19 (IgG2b, kappa). It has also been characterized for breast, oral, and colon cancers. In thisstudy, we investigated the antitumor activities of H2Mab-19 in pancreatic cancer xenograft models. We selected
MIA PaCa-2, a pancreatic cancer cell line which expresses HER2. H2Mab-19 showed high binding affinity(KD: 1.2 · 10-8 M) against MIA PaCa-2 cells. Furthermore, H2Mab-19 significantly reduced tumor development
in a MIA PaCa-2 xenograft model. These results suggest that treatment with H2Mab-19 may be a useful therapy
for patients with HER2-expressing pancreatic cancers.
Keywords: HER2, monoclonal antibody, antitumor activity

combination of trastuzumab and pertuzumab with chemotherapy has led to significant improvements in overall
survival.(13) Trastuzumab deruxtecan (DS-8201) comprises
three components, a novel enzyme-cleavable linker, and a
topoisomerase I inhibitor and exerts antitumor activity evenin low-HER2-expressing tumors.(14) DS-8201 has several
innovative features: (1) a high drug-to-antibody ratio, (2) atumor-selective cleavable linker, (3) a stable linker-payload
in circulation, and (4) a bystander effect.(15)In our recent studies, a novel anti-HER2 mAb (H2Mab-19;
IgG2b, kappa) was developed by immunizing mice with thepurified recombinant extracellular domain of HER2.(16)H2Mab-19 showed both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity(CDC) against BT-474 (a human breast cancer cell line) andHSC-2 or SAS (human oral cancer cell lines). Furthermore,
H2Mab-19 exerted antitumor activities in BT-474, HSC-2,
and SAS xenografts, respectively, suggesting that treatment
with H2Mab-19 may be a useful therapy for patients with
HER2-expressing breast and oral cancers. As HER2 has been
seen to be expressed in pancreatic cancers,(17,18) we herein
investigated antitumor activities in mouse xenograft modelsof pancreatic cancers.

Introduction

P

ancreatic cancer is the 11th most common cancer
worldwide, and there are an estimated 458,918 newcases and 432,242 deaths each year; it accounted for *4.5%of all cancer deaths in 2018.(1) Notably, pancreatic cancer isthe seventh leading cause of cancer in both males and females due to the poor prognosis and increasing age of the
population. Although surgery with adjuvant chemotherapy,
including gemcitabine, is the only viable choice to prolong
survival, only 10%–20% of patients with pancreatic cancer
have operable cancers at their initial diagnosis, and the recurrence rate remains very high.(2–5) Therefore, the development of new therapeutic approaches is urgently required to
improve the outcome of patients with pancreatic cancer.
Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in breast and gastric cancers
and is associated with poor clinical outcomes.(6–8) Trastuzumab and pertuzumab, which are humanized anti-HER2
monoclonal antibodies (mAbs), have been used in the treatment of HER2-positive breast cancer.(9–11) Treatment withtrastuzumab resulted in significantly improved survival
rates.(12) In comparison to trastuzumab monotherapy, the1
2
3

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Japan.

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Materials and Methods
Cell line

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MIA PaCa-2 was obtained from the Cell Resource Centerfor the Biomedical Research Institute of Development, Aging
and Cancer Tohoku University (Miyagi, Japan). MIA PaCa-2
was cultured in DMEM medium (Nacalai Tesque, Inc.,
Kyoto, Japan), supplemented with 10% heat-inactivated fetal
bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA),
100 U/mL of penicillin, 100 lg/mL of streptomycin, and0.25 lg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37C
in a humidified atmosphere containing 5% CO2 and 95% air.

KATO ET AL.

control mice, respectively. Additional antibodies were theninjected on days 6 and 14. Twenty days after cell implantation, all mice were euthanized by cervical dislocation, andtumor diameters and volumes were determined using amethod previously described.(19)
Statistical analyses

All data were expressed as mean – standard error of themean. Statistical analysis tests—ANOVA and Tukey–
Kramer’s test were performed using GraphPad Prism 6
software (GraphPad Software, Inc.). p < 0.05 was adopted as
the level of statistical significance.

Animals

Results

All animal experiments were performed by following
relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. Animal studies for antitumor activity were approved by the Institute of Microbial
Chemistry (Permit number: 2019-021). Mice were monitored
for health and weight every 2–4 days. The experiment was
conducted over three weeks. A bodyweight loss exceeding
25% and a maximum tumor size exceeding 3000 mm3 wereidentified as humane endpoints. Mice were euthanized bycervical dislocation, and the death was verified by respiratoryarrest and cardiac arrest.

Characterization of H2Mab-19 against a pancreaticcancer cell line

We first measured the surface expression of HER2
by human pancreatic cell line MIA PaCa-2. As expected,
H2Mab-19 recognized endogenous HER2 of MIA PaCa-2
cells by flow cytometry (Fig. 1A). We next examined theFlow cytometry

MIA PaCa-2 cells were harvested by being exposed briefly
to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid
(EDTA; Nacalai Tesque, Inc.). After washing with 0.1%
bovine serum albumin in phosphate-buffered saline (PBS),
MIA PaCa-2 cells were treated with 1 lg/mL anti-HER2(H2Mab-19) for 30 minutes at 4C and subsequently with
Alexa Fluor 488-conjugated anti-mouse IgG (1:1000; Cell
Signaling Technology, Inc., Danvers, MA). Fluorescencemicroscopy data were collected using an EC800 Cell Analyzer (Sony Corp., Tokyo, Japan).
Determination of the binding affinity

MIA PaCa-2 cells were suspended in 100 lL serially diluted H2Mab-19 (6 ng/mL–100 lg/mL), followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG (1:200;
Cell Signaling Technology, Inc.). Fluorescence microscopy
data were collected using an EC800 Cell Analyzer (SonyCorp.). The dissociation constant (KD) was obtained by fitting
binding isotherms to built-in one-site binding models using
GraphPad PRISM 6 software (GraphPad Software, Inc., La
Jolla, CA).
Antitumor activity of H2Mab-19 in the xenograftsof pancreatic cancers

Sixteen 6-week-old female BALB/c nude mice were purchased from Charles River and used when they were 7 weeks
old. MIA PaCa-2 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), and100 lL of this suspension (5 · 106 cells) was injected subcutaneously into the left flank. After day 1, 100 lg H2Mab-19
and control mouse IgG (Sigma-Aldrich Corp.) in 100 lL PBS
were given an intraperitoneal (i.p.) injection into treated and

FIG. 1. Characterization of H2Mab-19 using flow cytometry (A) MIA PaCa-2 cells were treated with H2Mab-19
and negative control (blocking buffer). (B) Determination ofbinding affinity of H2Mab-19 for MIA PaCa-2 cells using
flow cytometry.

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AN ANTI-HER2 MAB FOR PANCREATIC CANCERS

63

binding affinities (KD) of H2Mab-19 to MIA PaCa-2, whichshowed 1.2 · 10-8 M (Fig. 1B), indicating that H2Mab-19
shows high affinity to HER2-expressing pancreatic cancer
cell lines. These results suggest the possibility of targeting
HER2 as an antigen for immunotherapy.

Mice of day 20 are depicted in Figure 3A. The total bodyweight was not significantly different between the twogroups (Fig. 3B). These results indicate that H2Mab-19 exerts
a significant antitumor effect against HER2-expressing
pancreatic cancers.

Antitumor activity of H2Mab-19 in mouse xenograftsof pancreatic cancers

Discussion

Next, whether the cancer-cell surface binding of the
H2Mab-19 could induce cytotoxic activity against pancreaticcancer was investigated. MIA PaCa-2 cells were implanted
subcutaneously in the flanks of nude mice to study the
antitumor activity of H2Mab-19 on cell growth in vivo.
H2Mab-19 and control mouse IgG were injected (via i.p.
administration) three times, on days 1, 6, and 14 (after cell
injection) into treated and control mice, respectively. Tumor
formation was observed in mice in both H2Mab-19-treatedand control groups. H2Mab-19 treatment significantly reduced tumor development compared with tumor development
in control mice on days 14, 17, and 20 ( p < 0.01; Fig. 2A).
Resected tumors are depicted in Figure 2B. Weights of tumors from H2Mab-19-treated mice were significantly lessthan for tumors from IgG-treated control mice (Fig. 2C).

FIG. 2. Evaluation of the antitumor activity of H2Mab-19.
(A) Tumor volume was measured from MIA PaCa-2
xenografts. (B) Resected tumors of MIA PaCa-2 xenografts(day 20). (C) Tumor weight was measured from MIA PaCa2 xenografts. Values are mean – SEM. **p < 0.01 calculated
from the Tukey–Kramer’s test. SEM, standard error of themean.

Antibody-based cancer immunotherapies that target
tumor-associated antigens and trigger a patient’s immune
system to attack cancer cells have been developed in thepast two decades and have become one of the most promising strategies to treat cancer patients.(20) The majorityof antibody-based immunotherapies relies on antibodymediated recognition of cancer cell surface antigens that areeither overexpressed or selectively expressed, compared withhealthy cells, to recruit immune effector functions, such as
ADCC and CDC.(20,21)
We previously developed an anti-HER2 mAbs, H2Mab119(18) using CasMab technology.(22) H2Mab-119 is usefulfor flow cytometry, western blot, and immunohistochemical
analyses. The subclass of H2Mab-119 was determined to bemouse IgG1; therefore, H2Mab-119 does not possess ADCC
or CDC. We further tried to develop an anti-HER2 mAbof IgG2b subclasses using CasMab technology as mouse IgG2b
antibodies show ADCC and CDC activity.(23) A novel antiHER2 mAb (H2Mab-19) of IgG2b was recently established.(16)

FIG. 3. Body weights of the mice with the MIA PaCa-2
xenografts. (A) The appearance of treated mice on day 20.
(B) Body weights of the mice with the MIA PaCa-2 xenografts were measured for 20 days. n.s., not significant.

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64

KATO ET AL.

H2Mab-19 possesses both ADCC and CDC against humanbreast cancer or human oral cancer cell lines. Furthermore,
H2Mab-19 exerted antitumor activities in breast cancer or oral
cancer xenografts. We, therefore, investigated whether H2Mab19 demonstrates antitumor activities in mouse xenograftmodels of MIA PaCa-2 as HER2 has been reported to be expressed in pancreatic cancers.(17,18)
Antigen-antibody affinity is thought to be an importantfactor influencing the outcome of antibody-based therapy.(24)The binding affinity (KD) of H2Mab-19 to MIA PaCa-2 wasdetermined to be 1.2 · 10-8 M using flow cytometry, indicating that H2Mab-19 shows high affinity to HER2expressing pancreatic cell lines similarly with the KD of
H2Mab-19 to BT-474 (2.3 · 10-8 M), HSC-2 (9.5 · 10-9 M),
and SAS (5.5 · 10-9 M).(16)In this study, we selected a MIA PaCa-2 cell line for in vivo
investigation, which was detected by H2Mab-19 in flow cytometry (Fig. 1A). In our previous study, H2Mab-19 treatment significantly reduced tumor development of breast andoral cancer xenograft models compared with development incontrol mice.(16) H2Mab-19 also exerted the antitumor activity against MIA PaCa-2 xenografts (Fig. 2). Further studies
using the other pancreatic cancer xenografts should be performed in the future to confirm that H2Mab-19 could be auseful therapy for patients with HER2-expressing pancreaticcancers.
Acknowledgments

We thank Takuro Nakamura and Akiko Harakawa fortechnical assistance.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by AMED underGrant Numbers: JP20am0401013 (Y.K.), JP20am0101078(Y.K.), and JP20ae0101028 (Y.K.), and by JSPS KAKENHI
Grant Number 17K07299 (M.K.K.) and Grant Number
19K07705 (Y.K.).
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi
Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 14, 2020
Accepted: April 19, 2020