MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 4, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0009

An Anti-Human Epidermal Growth Factor Receptor 2
Monoclonal Antibody H2Mab-19 Exerts Antitumor Activity
in Mouse Colon Cancer Xenografts

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

Trastuzumab is a humanized antibody against human epidermal growth factor receptor 2 (HER2) that offers
significant survival benefits to patients with HER2-overexpressing breast or gastric cancer. HER2 is also known
to be overexpressed in colon cancers. In this study, a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19(IgG2b, k) was characterized for its anticancer activity in colon cancers. H2Mab-19 showed both antibodydependent cellular cytotoxicity and complement-dependent cytotoxicity activities against Caco-2, a coloncancer cell line. Furthermore, H2Mab-19 significantly reduced tumor development in a Caco-2 xenograft model.
These results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing
colon cancers.
Keywords: HER2, monoclonal antibody, antitumor activity

IntroductionHumanized antibody against human epidermal growth
factor receptor 2 (HER2) monoclonal antibodies (mAbs)(e.g., trastuzumab and pertuzumab) have been used for thetreatment of HER2-positive breast cancer.(1–3) Treatment
with trastuzumab results in significant survival benefits forthese patients.(4) In comparison with trastuzumab monotherapy, the combination of trastuzumab plus pertuzumab
and chemotherapy has led to significant improvements inoverall survival.(5) Trastuzumab deruxtecan (DS-8201),
which is a mAb that combines a novel enzyme-cleavable
linker and a topoisomerase I inhibitor, was recently developed.(6) Importantly, DS-8201 shows antitumor activity in
low-HER2-expressing tumors, DS-8201 has several innovative features: (i) a highly potent novel payload with a highdrug-to-antibody ratio, (ii) good homogeneity, (iii) a tumorselective cleavable linker, (iv) a stable linker-payload incirculation, (v) a cytotoxic agent with a short in vivo half-life,and (vi) bystander effect.(7)We recently developed a novel anti-HER2 mAb (H2Mab19; IgG2b, k) by immunizing BALB/c mice with purified
recombinant protein corresponding to the extracellulardomain of HER2.(8) H2Mab-19 demonstrated both antibodydependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC) against a human breast
1
2
3

cancer cell line (BT-474) as well as two human oral cancer
cell lines (HSC-2 and SAS). Furthermore, H2Mab-19 significantly reduced tumor development in BT-474, HSC-2,
and SAS xenografts. These results suggest that treatment
with H2Mab-19 may be a useful therapy for patients with
HER2-expressing breast and oral cancers. HER2 has also
been reported to be expressed in colorectal cancers.(9)Based on the antitumor activities of H2Mab-19 in breastand oral cancer cell lines, we sought to investigate whether
H2Mab-19 displayed similar ADCC and CDC activities
in vitro in a human colon cancer cell line, and to examine its
antitumor activity in vivo in a mouse xenograft model ofcolon cancer.
Materials and Methods
Cell lines

The LS 174T, Caco-2, HCT-116, HT-29, COLO 201,
HCT-8, and SW1116 cell lines were obtained from theAmerican Type Culture Collection (Manassas, VA), and theHCT-15, DLD-1, and COLO 205 cell lines were obtained
from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer Tohoku University (Miyagi, Japan). COLO 201, SW1116, HCT-15,
DLD-1, and COLO 205 were cultured in RPMI 1640 medium

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, Shizuoka, Japan.

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(Nacalai Tesque, Inc., Kyoto, Japan), and LS 174T, Caco-2,
HCT-116, HT-29, and HCT-8 were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) medium (Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated fetal
bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham,
MA, USA), 100 U/mL of penicillin, 100 lg/mL of streptomycin, and 25 lg/mL of amphotericin B (Nacalai Tesque,Inc.). All cell lines were incubated at 37C in a humidified
atmosphere containing 5% CO2 and 95% air.

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Animals

All animal experiments were performed in accordancewith relevant guidelines and regulations to minimize animal
suffering and distress in the laboratory. Animal studies for
ADCC were approved by the institutional committee forexperiments of the Institute of Microbial Chemistry (Permit
No.: 2019-066). Animal studies for antitumor activity wereapproved by the institutional committee for experiments of
the Institute of Microbial Chemistry (Permit No.: 2019-004).
Mice were monitored for health and weight every 2–5 days.
Experiment duration was 3 weeks. A bodyweight loss >25%
and a maximum tumor size >3000 mm3 were identified as
humane endpoints. Mice were euthanized by cervical dislocation, and the death was verified by respiratory arrest andcardiac arrest.
Flow cytometry

Cells were harvested by brief exposure 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), cells weretreated with 1 lg/mL anti-HER2 mAb (H2Mab-19) for 30
minutes at 4C, followed by incubation with Alexa Fluor
488-conjugated anti-mouse IgG (1:1000; Cell Signaling
Technology, Inc., Danvers, MA, USA). Fluorescence data
were collected using an EC800 Cell Analyzer (Sony Corp.,
Tokyo, Japan).
Determination of the binding affinity

Cells were suspended in 100 lL of serially diluted
H2Mab-19 (6 ng/mL to 100 lg/mL), followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG(1:200; Cell Signaling Technology, Inc.). Fluorescencedata were collected using an EC800 Cell Analyzer (SonyCorp.). The dissociation constant (KD) was obtained byfitting binding isotherms to built-in one-site binding
models in GraphPad PRISM 6 (GraphPad Software, Inc.,
La Jolla, CA, USA).

KATO ET AL.

AM (Thermo Fisher Scientific, Inc.) and resuspended in thesame medium. The target cells were plated in 96-wellplates (2 · 104 cells/well) and mixed with effector cells,
100 lg/mL of anti-HER2 antibodies, or control IgG (mouse
IgG2b) (Sigma-Aldrich Corp., St. Louis, MO, USA). After a
4-hour incubation, the Calcein AM release of supernatantfrom each well was measured. Fluorescence intensity wasdetermined using a microplate reader (Power Scan HT;
BioTek Instruments, Winooski, VT, USA) with an excitation wavelength of 485 nm and an emission wavelength of
538 nm. Cytolytic activity (% of lysis) was calculated as% lysis = (E - S)/(M - S) · 100, where E is fluorescence ofcombined target and effector cells, S is spontaneous fluorescence of target cells only, and M is maximum fluorescence measured after lysing 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

Cells were plated in 96-well plates (2 · 104 cells/well), in
DMEM supplemented with 10% FBS, and incubated for
5 hours at 37C with 100 lg/mL of anti-HER2 antibodies orcontrol IgG (mouse IgG2b) (Sigma-Aldrich Corp.) and 10%of rabbit complement (Low-Tox-M Rabbit Complement;
Cedarlane Laboratories, Hornby, Ontario, Canada). To assess
cell viability, an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;
inner salt] assay was performed using a CellTiter 96 AQueous assay kit (Promega, Madison, WI, USA).
Antitumor activity of H2Mab-19 in the xenograftsof colon cancers

Sixteen 6-week-old female BALB/c nude mice werepurchased from Charles River and used at 7 weeks of age.
Caco-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, USA).
Mice were injected subcutaneously into the left flank(100 lL) with the aforementioned suspension (5 · 106
cells). After day 1, 100 lg H2Mab-19 or control mouse IgG(Sigma-Aldrich Corp.) in 100 lL PBS were injected into
treated and control mice through intraperitoneal injection(i.p.), respectively. Additional antibodies were then injected on days 7 and 14. Eighteen days after cell implantation, all mice were euthanized by cervical dislocation,and tumor diameters and volumes were determined aspreviously described.(10)
Statistical analyses

Antibody-dependent cellular cytotoxicity

Six 6-week-old female BALB/c nude mice were purchasedfrom Charles River (Kanagawa, Japan). After euthanization
by cervical dislocation, their spleens were removed aseptically and single-cell suspensions were obtained by forcing
spleen tissues through a sterile cell strainer (352360, BD
Falcon; Corning, New York, NY, USA) using a syringe.
Erythrocytes were lysed with a 10-second exposure to icecold distilled water. Splenocytes were washed with DMEM
and resuspended in DMEM with 10% FBS and used as effector cells. Target cells were labeled with 10 lg/mL Calcein

All data were expressed as mean – standard error of themean. Statistical analysis used ANOVA and Tukey–Kramer’s
test with R statistical p < 0.05 was adopted as a level of statistical significance.
Results
Characterization of H2Mab-19
against colon cancer cell lines

H2Mab-19 recognized endogenous HER2 in the 10 coloncancer cell lines tested, HCT-15, LS 174T, DLD-1, Caco-2,

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AN ANTI-HER2 MAB FOR COLON CANCERS

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FIG. 1. Characterization of H2Mab-19 using flow cytometry. (A) Colon cancer cell lines were treated with H2Mab-19.
The black line denotes the negative control (blocking buffer). (B) Determination of the binding affinity of H2Mab-19 forCaco-2 cells using flow cytometry.

HCT-116, HT-29, COLO 201, COLO 205, HCT-8, andSW1116, which are HER2-positive(11) (Fig. 1A). Using flow
cytometry, the binding affinity (KD) of H2Mab-19 to the
Caco-2 cell line was 1.1 · 10-8 M, indicating that H2Mab-19
shows a high affinity to this HER2-expressing colon cancer
cell line.
H2Mab-19-induced ADCC and CDC activities
in a colon cancer cell line

This study examined whether H2Mab-19 induced ADCC
and CDC in the HER2-expressing colon cancer cell line,
Caco-2. H2Mab-19 is a mouse IgG2b subclass antibody,which possesses both ADCC and CDC activities.(8) As
expected, H2Mab-19 exhibited high ADCC activity (32.6%
of cytotoxicity; Fig. 2A) and high CDC activity (50.7% of
cytotoxicity; Fig. 2B) against Caco-2 cells, suggesting that

H2Mab-19 might exert antitumor activity in an in vivo
model of colon cancer.
Antitumor activity of H2Mab-19 in mouse
xenografts of colon cancer

To study the antitumor activity of H2Mab-19 on cell
growth in vivo, Caco-2 cells were implanted subcutaneously
in the flanks of nude mice. H2Mab-19 or control mouse IgG
was injected i.p. three times on days 1, 7, and 14 after Caco-2cell injection into treated and control mice, respectively.
Tumor formation was observed in both H2Mab-19-treatedand control groups. H2Mab-19 treatment significantly reduced tumor development compared with control mice ondays 5, 7, 12, 15, and 18 of observation (Fig. 3A). Resected
tumors are shown in Figure 3B. The weight of the tumors
excised from H2Mab-19-treated mice was significantly less

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126

FIG. 2. ADCC and CDC activities. (A) ADCC activity
against Caco-2 cells. (B) CDC activity against Caco-2 cells.
An asterisk indicates statistical significance (**p < 0.01, n.s.,
not significant, Tukey–Kramer’s test). ADCC, antibodydependent cellular cytotoxicity; CDC, complement-dependent
cytotoxicity.

than for tumors from IgG-treated control mice (Fig. 3C).
Total body weight was not significantly different between thetwo groups (Fig. 4).

KATO ET AL.

FIG. 3. Evaluation of antitumor activity of H2Mab-19.
(A) Tumor volume was measured in mice with Caco-2 xenografts. (B) Resected tumors of Caco-2 xenografts on day
18. (C) Tumor weight was measured from excised Caco-2
xenografts. Values are mean – SEM. **p < 0.01, the Tukey–
Kramer’s test. SEM, standard error of the mean.

Discussion

We previously developed several anti-HER2 mAbs, including H2Mab-77,(12) H2Mab-119,(13) and H2Mab-139(11)using CasMab technology.(14) For this technology, it is critical that immunogens are produced using cancer cell lines,
such as LN229 glioblastoma cells, which express cancerspecific glycan-attached membrane proteins. These antibodies are useful for flow cytometry, western blot, andimmunohistochemical analyses. Unfortunately, the subclassof these mAbs was determined to be mouse IgG1; therefore,
they do not possess ADCC or CDC activities. Consequently,
we further tried to develop an anti-HER2 mAb of either the
IgG2a or IgG2b subclass using CasMab technology because
both IgG2a(15) and IgG2b antibodies(16) show ADCC and CDC
activity. We successfully established a novel anti-HER2
mAb (H2Mab-19) of subclass IgG2b.(8) H2Mab-19 possesses
both ADCC and CDC activity in both human breast cancer

FIG. 4. Body weights of the mice with the Caco-2 xenografts. Body weights of the mice with the Caco-2 xenografts
were measured for 18 days. n.s., not significant.

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AN ANTI-HER2 MAB FOR COLON CANCERS

and oral cancer cell lines. Furthermore, H2Mab-19 exerted
antitumor activity in human breast cancer and oral cancer
xenografts. HER2 has been reported to be expressed in a
number of colorectal cancers.(9) Based on this data, we sought
to investigate whether H2Mab-19 has ADCC and CDC activities in vitro in a human colon cancer cell line as well as
determine its antitumor activity in a mouse xenograft modelof colon cancer.
Using flow cytometric analysis, the binding affinity (KD) of
H2Mab-19 to Caco-2 was determined to be 1.1 · 10-8 M.
These results were similar to the KD of H2Mab-19 observed in
BT-474 (2.3 · 10-8 M), HSC-2 (9.5 · 10-9 M), and SAS(5.5 · 10-9 M) cell lines.(8) In addition, H2Mab-19 exhibitedhigh ADCC activity (32.6% of cytotoxicity; Fig. 2A) and high
CDC activity (50.7% of cytotoxicity; Fig. 2B) against Caco-2
cells, which are compatible with the previous data obtained of
ADCC and CDC activities of H2Mab-19 against human breast
cancer and oral cancer cell lines: ADCC activities of H2Mab19 against BT-474, HSC-2, and SAS were 31.5%, 26.2%,
and 38.1%, respectively; CDC activities of H2Mab-19 against
BT-474, HSC-2, and SAS were 45.3%, 50.2%, and 52.3%,
respectively,(8) indicating that H2Mab-19 might exert highcytotoxicity against a colon cancer xenograft model.
In this study, we selected the Caco-2 cell line from 10
HER2-expressing colon cancer cell lines, which were detected by the H2Mab-19 in flow cytometry (Fig. 1A), for thecolon cancer xenograft model because we previously established Caco-2 xenograft models.(17) In our previous
study, H2Mab-19 treatment significantly reduced tumor
development in both breast and oral cancer xenografts
compared with tumor development in control mice.(8) Similar to our previous study, we found that H2Mab-19
demonstrated antitumor activity against Caco-2 xenografts(Fig. 3). Further studies using the other colon cancer xenografts should be performed in the future to confirm that
H2Mab-19 could be a useful therapy for patients with
HER2-expressing colon cancers.
Acknowledgments

We thank Ms. Akiko Harakawa for technical assistance ofanimal experiments.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by AMED under Grant
Numbers JP20am0401013 (Y.K.), JP20am0101078 (Y.K.),
and JP20ae0101028 (Y.K.), and by JSPS KAKENHI Grant
Numbers 17K07299 (M.K.K.) and 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
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 11, 2020
Accepted: May 26, 2020