ONCOLOGY REPORTS 46: 173, 2021

Anti‑HER3 monoclonal antibody exerts antitumor activity
in a mouse model of colorectal adenocarcinoma
TEIZO ASANO1*, TOMOKAZU OHISHI2*, JUNKO TAKEI1,3, TAKURO NAKAMURA1,REN NANAMIYA1, HIDEKI HOSONO1, TOMOHIRO TANAKA1, MASATO SANO1,
HIROYUKI HARADA3, MANABU KAWADA2, MIKA K. KANEKO1 and YUKINARI KATO1,4
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai,
Miyagi 980‑8575; 2Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
Numazu‑shi, Shizuoka 410‑0301; 3Department of Oral and Maxillofacial Surgery, Graduate School of
Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo‑ku, Tokyo 113‑8510;
4Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980‑8575, Japan
Received March 2, 2021; Accepted May 21, 2021
DOI: 10.3892/or.2021.8124
Abstract. HER3 belongs to the epidermal growth factor receptor(EGFR) family and is known to form an active heterodimer withother three family members EGFR, HER2, and HER4. HER3
is overexpressed in lung, breast, colon, prostate, and gastriccancers. In the present study, we developed and validated ananti‑HER3 monoclonal antibody (mAb), H3Mab‑17 (IgG2a,kappa), by immunizing mice with HER3‑overexpressed
CHO‑K1 cells (CHO/HER3). H3Mab‑17 was found to react
specifically with endogenous HER3 in colorectal carcinoma
cell lines, using flow cytometry. The K D for H3Mab‑17 in
CHO/HER3 and Caco‑2 (a colon cancer cell line) were deter‑
mined to be 3.0x10 ‑9 M and 1.5x10 ‑9 M via flow cytometry,
respectively, suggesting high binding affinity of H3Mab‑17 to
HER3. Then, we assessed the H3Mab‑17 antibody‑dependent
cellular cytotoxicity (ADCC) and complement‑dependent
cytotoxicity (CDC) against Caco‑2, and evaluated its antitumor
capacity in a Caco‑2 xenograft model. In vitro experiments

Correspondence to: Professor Yukinari Kato, Department of
Molecular Pharmacology, Tohoku University Graduate School of
Medicine, 2‑1 Seiryo‑machi, Aoba‑ku, Sendai, Miyagi 980‑8575,
Japan
E‑mail: yukinarikato@med.tohoku.ac.jp*

Contributed equally

Abbreviations: ADC, antibody‑drug conjugate; ADCC,
antibody‑dependent cellular cytotoxicity; BSA, bovine serum
albumin; CBIS, Cell‑Based Immunization and Screening; CDC,
complement‑dependent cytotoxicity; DMEM, Dulbecco's modified
Eagle's medium; EDTA, ethylenediaminetetraacetic acid; FBS, fetal
bovine serum; mAb, monoclonal antibody; PBS, phosphate‑bufferedsaline; SEM, standard error of the mean
Key words: HER3, monoclonal antibody, antibody‑dependent
cellular cytotoxicity, complement‑dependent cytotoxicity, antitumor
activity, colorectal cancer

revealed H3Mab‑17 had strongly induced both ADCC and CDC
against Caco‑2 cells. In vivo experiments on Caco‑2 xenografts
revealed that H3Mab‑17 treatment significantly reduced tumor
growth compared with the control mouse IgG. These data indi‑cated that H3Mab‑17 could be a promising treatment option for
HER3‑expressing colon cancers.
Introduction
The epidermal growth factor receptor (EGFR) family, also
known as HER or ErbB, has a tyrosine kinase domain in
its intracellular region (1). The EGFR family transduces
extracellular to intracellular signals through the activationof tyrosine kinase domain (1). By binding to the ligand, theextracellular domain promotes the formation of homodimers
or heterodimers between the EGFR family receptors (2,3).
This dimerization is essential for the activation of the tyrosine
kinase domain and intracellular signaling pathways such as
Ras/MAPK, PI3K/Akt, and JAK/STAT (4,5).
The EGFR family consists of four members [EGFR(HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4
(ErbB4)] and each member has different ligands: EGFR binds
to seven ligands such as EGF, TGF‑α, and epigen; HER3 binds
to neuregulin1 and neuregulin2; HER4 binds to seven ligands
such as heparin binding‑EGF, betacellulin, and epiregulin. In
contrast, there is no ligand for HER2 (6,7). Although HER3 hasa tyrosine kinase domain, its kinase activity is impaired (8,9).
Therefore, transphosphorylation by other members of the
EGFR family is required to activate HER3. HER3 can form anactive heterodimer with the other three members of the EGFR
family (2,10‑13).
The EGFR family plays an essential role in regulating cell
growth and in the differentiation, proliferation, and survival of
normal cells. Insufficient EGFR signaling is associated with
Alzheimer's disease and multiple sclerosis (14), while the over‑expression of EGFR family is associated with the developmentof tumors (15‑17). The EGFR family has been found to be over‑expressed in many cancers as below: EGFR in breast, non‑small

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ASANO et al: ANTITUMOR ACTIVITIES OF AN ANTI-HER3 mAb

cell lung, and prostate cancers (18); HER2 in breast, colon,lung, and pancreatic cancers (18); HER3 in lung, breast, colon,
prostate, and stomach cancers (2,19); HER4 in non‑small celllung, and ovarian cancers (20,21). Therefore, the EGFR familyis thought to be a valid target for candidates in cancer therapy.
High expression of HER3 is thought to be an established nega‑tive prognostic factor in several solid tumors including colorectal
cancer (22,23). Metastatic colorectal cancer is one of the mostaggressive tumors, associated with high mortality rates world‑
wide (24). In a previous study, 79% of primary tumors were foundto present a high HER3 expression and there was a correlation
between HER3 expression in primary tumors and corresponding
lymph node metastases in 236 colorectal cancer patients (25).
In addition, elevated HER3 expression was associated withshorter overall survival and disease‑free survival in patients withcolorectal cancer (25). Furthermore, HER3 downregulation incolorectal cancer cell lines caused G2‑M cell‑cycle arrest, leading
to apoptosis and abrogated cell proliferation, migration, and inva‑
sion (22). Altogether, these results suggest that HER3 can be apotential therapeutic target for colorectal cancer.
Several monoclonal antibodies (mAbs) have been
established as an innovative immunotherapy against
tumors. Programmed cell death 1 (PD‑1) and cytotoxic
T‑lymphocyte‑associated protein 4 (CTLA‑4) are inhibitory
receptors for immune checkpoints, which are expressed on
the surface of T cells (26‑29). Anti‑PD‑1 and anti‑CTLA‑4
mAbs have been reported as potential anticancer drugs (30,31).
Nivolumab and pembrolizumab are anti‑PD‑1 mAbs, whichwere approved by the US Food and Drug Administration (32‑34).
Both mAbs activate the immune system to attack tumors, byblocking the interaction between PD‑1 and its ligand, PD‑L1,
which is expressed in cancer cells. DNA mismatch repair
deficient tumors have very high levels of DNA microsatellite
instability, and it is known that microsatellite instable tumors
have highly upregulated expression of multiple immune check‑point proteins, including PD‑1 compared with microsatellite
stable tumors; therefore, nivolumab and pembrolizumab are
available for the treatment of DNA mismatch repair deficiency
and microsatellite instable subset of colorectal cancer (35‑37).
Several antibody drugs have been developed against
ligands, such as transforming growth factor (TGF)‑ α and
EGF, or receptors, such as EGFR (38). These mAbs neutralize
the interaction between ligands and receptors. Antibody‑drug
conjugate (ADC) is a complex molecule, which is composedof an antibody, linker, and an anticancer drug, and delivers
the anticancer drug to target cells (39). Moreover, some mAbs
possess antibody‑dependent cellular cytotoxicity (ADCC)
or complement‑dependent cytotoxicity (CDC). Cetuximab,a mouse/human chimeric IgG1 against EGFR, binds to theligand‑binding site of EGFR, and inhibits the activation anddimerization of EGFR (38). Cetuximab has been used for thetreatment of metastatic colorectal cancer, metastatic non‑small
cell lung cancer, and head and neck squamous cell carcinomas(HNSCC) (38). Trastuzumab, a humanized mAb against
HER2, has been used to treat HER2‑positive cancers, such asbreast cancers and gastric cancers (40,41). Trastuzumab binds
to the extracellular domain of HER2 and downregulates activa‑tion of AKT (42). Moreover, trastuzumab exhibited ADCC in amouse model (43). However, it has been shown that some typesof cancers are resistant to cetuximab and trastuzumab (44,45).

Most HNSCCs are resistant to cetuximab, because
cetuximab treatment induces HER2/HER3 dimerizationand HER3 activation in HNSCC cell lines (44). It has beenreported that anti‑HER3-ADC exerts antitumor effect onbreast cancer cells, which have resistance to trastuzumab (45).
For this reason, the development of anti‑HER3 mAbs has
been required for cancer therapy. Seribantumab and lumretu‑
zumab are anti‑HER3 mAbs, which block HER3‑neuregulininteraction and inhibit HER3 heterodimerization and phos‑
phorylation (46,47). Lumretuzumab is also known to have
ADCC activity (46). Phase II and phase Ib/II trial are now
ongoing concerning seribantumab and lumretuzumab, respec‑tively (47,48). An anti‑HER3-ADC (U3‑1402), composedof an anti‑HER3 mAb (patritumab) and a novel topoisom‑erase I inhibitor (DX‑8951 derivative; DXd) has enteredphase I and II trials for the treatment of HER3‑positive
non‑small cell lung cancers (NCT04676477), metastatic
breast cancers (NCT02980341), and colorectal cancers(NCT04479436) (49‑51). Preliminary results demonstrate thatU3‑1402 treatment appears to be safe and exhibits antitumor
activity, suggesting that HER3‑targeting therapy may be effec‑
tive for HER3‑overexpressing metastatic breast cancers (50).
It has been reported that one amino acid substitution in
EGFR in tumors causes acquisition of resistance to gefitinib
after gefitinib treatment (52,53); therefore, HER3 may alsoacquire resistance to seribantumab and lumretuzumab in the
future. To characterize the HER3 and HER3‑targeting cancer
therapy, the development of further anti‑HER3 specific mAbs
is required. In this study, we developed a novel anti‑HER3 mAb
against colon cancers using a Cell‑Based Immunization andScreening (CBIS) method (54). Furthermore, we investigatedwhether a novel anti‑HER3 mAb shows ADCC/CDC activities
or antitumor activities for colon cancers.
Materials and methods
Construction of plasmids. The Genome Network Project clone
IRAK174J18 (HER3) was provided by the RIKEN BioResource
Research Center through the National BioResource Project of
the MEXT and AMED agencies of Japan. HER3 DNA plus
N‑terminal PA16 tag, recognized by NZ‑1, was subcloned intoa pCAG‑Ble vector (FUJIFILM Wako Pure Chemical Corp.)
and named pCAG/PA16‑HER3. HER3 DNA plus C‑terminal
PA tag, recognized by NZ‑1, was subcloned into a pCAG‑Neo
vector (FUJIFILM Wako Pure Chemical Corp.) and named
pCAG/HER3‑PA.
Cell lines. A mouse myeloma cell line (P3X63Ag8U.1; P3U1),
Chinese hamster ovary (CHO)‑K1 cells, a glioblastoma cellline (LN229), colorectal adenocarcinoma cell lines (Caco‑2,
LS 174T, COLO 201, HCT‑8, SW1116, and HT‑29), and acolorectal carcinoma cell line (HCT 116) were obtained
from the American Type Culture Collection. Colon adeno‑carcinoma cell lines (HCT‑15, COLO 205, and DLD‑1) and abreast adenocarcinoma cell line (MCF7) were obtained fromthe Cell Resource Center for Biomedical Research Instituteof Development, Aging and Cancer at Tohoku University.
CHO/PA16‑HER3 and CHO/HER3‑PA were established bytransfecting pCAG/PA16‑HER3 and pCAG/HER3‑PA, respec‑tively, into CHO‑K1 cells using the Neon Transfection System

ONCOLOGY REPORTS 46: 173, 2021

(Thermo Fisher Scientific, Inc.). A few days after transfection,
cells positive for anti‑HER3 mAb (clone D22C5; cat. no. 12708;
Cell Signaling Technology, Inc.) were sorted using a cell
sorter (SH800; Sony Biotechnology Corp.). CHO/mock (Ble)and CHO/mock (Neo) were established by transfection of thepCAG‑Ble vector and pCAG‑Neo vector, respectively. Stabletransfectants of CHO/mock (Ble) and CHO/PA16‑HER3
were cultured at 37˚C for 14 days on media containing
0.5 mg/ml of Zeocin (InvivoGen), and stable transfectants of
CHO/mock (Neo) and CHO/HER3‑PA were cultured at 37˚C
for 14 days on media containing 0.5 mg/ml of G418 (Nacalai
Tesque, Inc.). BINDS‑30 [MCF7/HER3‑knockout (KO)cells] were produced using CRISPR/Cas9 plasmids
targeting human HER3 (http://www.med‑tohoku‑antibody.
com/topics/001_paper_cell.htm). Using TruGuide gRNA
tool, gRNA of HER3/ERBB3 (NM_001005915) was selectedfrom GeneArt predesigned gRNAs database (Thermo Fisher
Scientific, Inc.). gRNA sequence used was GTC​CCG​TGA​GCA​CAA​T CT​CA(agg), which targeted exon 3 of HER3
(Assay ID: CRISPR764358). Double strand gRNA sequence
was subcloned into GeneArt CRISPR Nuclease Vector withOFP Reporter (Thermo Fisher Scientific, Inc.). P3U1, CHO‑K1,
CHO/PA16‑HER3, CHO/HER3‑PA, COLO 201, COLO 205,
SW1116, DLD‑1, MCF7, and BINDS‑30 were cultured in
Roswell Park Memorial Institute (RPMI)‑1640 media (Nacalai
Tesque, Inc.). LN229, Caco‑2, HCT 116, HCT‑15, HT‑29, LS
174T, and HCT‑8 were cultured in Dulbecco's modified Eagle's
medium (DMEM; Nacalai Tesque, Inc.). RPMI‑1640 and
DMEM were supplemented with 10% heat‑inactivated fetal
bovine serum (FBS; Thermo Fisher Scientific Inc.), 100 U/mlof penicillin (Nacalai Tesque, Inc.), 100 µg/ml streptomycin(Nacalai Tesque, Inc.), and 0.25 µg/ml amphotericin B(Nacalai Tesque, Inc.), and incubated at 37˚C in a humidified
atmosphere containing 5% CO2.
Preparation of the purified antibodies. Purified mouse
IgG (cat. no. I8765) and mouse IgG2a (cat. no. M7769)
were purchased from Sigma‑Aldrich; Merck KGaA. An
anti‑HER3 mAb was purified using Protein G‑Sepharose(GE Healthcare BioSciences).
Hybridoma production. Female BALB/c mice (6 weeks old)
were purchased from CLEA Japan and kept under specificpathogen‑free conditions. All animal experiments wereconducted in accordance with the relevant guidelines andregulations in order to minimize animal suffering and distressin the laboratory. The Animal Care and Use Committee of
Tohoku University approved all the animal experiments(permit no. 2019NiA‑001). Mice were euthanized by cervicaldislocation under inhalation anesthesia using 2% of isoflurane
for both induction and maintenance, and the death was verified
to be respiratory and cardiac arrest.
CBIS method was used as previously reported (54) to develop
mAbs against HER3 (Fig. 1). Two eight‑week‑old BALB/cfemale mice were intraperitoneally (i.p.) immunized with
CHO/PA16‑HER3 cells (1x108) along with Imject Alum adju‑
vant (Thermo Fisher Scientific, Inc.) (Fig. 1A). The procedureincluded three additional immunizations, followed by a final
booster injection administered i.p. two days before the spleencell harvesting. Spleen cells were then fused with P3U1 cells

3using PEG1500 (Roche Diagnostics) (Fig. 1B). The developed
hybridomas were seeded into 96‑well plates, and hybridomas
were grown at 37˚C for 10 days in RPMI‑1640 media with HAT
Supplement (50X) (cat. no. 21060017; Thermo Fisher Scientific,Inc.) for selection. Supernatants positive for CHO/HER3‑PA andnegative for CHO‑K1 were selected by flow cytometry (Fig. 1C).
After limiting dilution, supernatants positive for LN229 wereselected by flow cytometry. Finally, anti‑HER3 mAb‑producing
hybridomas were established (Fig. 1D).
Western blot analysis. Cell pellets were resuspended inphosphate‑buffered saline (PBS; Nacalai Tesque, Inc.) with
1% Triton X‑100 (cat. no. 168‑11805; FUJIFILM Wako Pure
Chemical Corp.) and 50 µg/ml aprotinin (product no. 03346‑84;
Nacalai Tesque, Inc.). Cell debris was removed by centrifugation
at 21,880 x g for 10 min at 4˚C. Protein concentration was deter‑
mined by BCA method. Cell lysates (10 µg) were boiled in sodium
dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.).
Proteins were electrophoresed on 5‑20% polyacrylamide gels
(FUJIFILM Wako Pure Chemical Corp.) and transferred onto
polyvinylidene difluoride (PVDF) membranes (Merck KGaA).
After blocking with 4% skim milk (Nacalai Tesque, Inc.) at room
temperature for 30 min, PVDF membranes were incubated with ananti‑HER3 mAb (diluted 1:1,000; clone D22C5) and anti‑β‑actinmAb (1 µg/ml; clone AC‑15; cat. no. A1978; Sigma‑Aldrich;
Merck KGaA) at room temperature for 30 min, followed by incu‑
bation with peroxidase‑conjugated anti‑rabbit immunoglobulins(diluted 1:1,000; cat. no. P0448; Agilent Technologies Inc.) andperoxidase‑conjugated anti‑mouse immunoglobulins (diluted
1:1,000; cat. no. P0260; Agilent Technologies Inc.), respec‑tively, at room temperature for 30 min. Blots were developed
using ImmunoStar LD (cat. no. 290‑69904; FUJIFILM Wako
Pure Chemical Corp.) or Pierce™ ECL Plus Western Blotting
Substrate (cat. no. 32132; Thermo Fisher Scientific, Inc.) andimaged with a Sayaca‑Imager (DRC Co., Ltd.). Qcapture Pro
software (DRC Co., Ltd) was used for the densitometry.
Flow cytometry analyses. Cells (2x10 5 cells/ml) were
harvested after brief exposure to 0.25% trypsin in 1 mM
ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque,Inc.). After being washed with 0.1% bovine serum albumin(BSA, Nacalai Tesque, Inc.) in PBS, cells were treatedwith 1 µg/ml of anti‑HER3 mAbs, for 30 min at 4˚C, andwith Alexa Fluor 488‑conjugated anti‑mouse IgG (1:1,000;
cat. no. 4408; Cell Signaling Technology, Inc.). Fluorescencedata were collected using a flow cytometer: the EC800 Cell
Analyzer (Sony Biotechnology Corp.).
Determination of the binding affinity. Cells (2x105 cells/ml)
were suspended in 100 µl of serially diluted anti‑HER3 mAb(6 ng/m l‑25 µg/m l), followed by the addition of
Alexa Fluor 488‑conjugated anti‑mouse IgG (1:200).
Fluorescence data were collected using a flow cytometer: The
BD FACSLyric (BD Biosciences). The dissociation constant (KD)was calculated by fitting binding isotherms to built‑in one‑sitebinding models in GraphPad Prism 8 (GraphPad Software, Inc.).
ADCC activity of an anti‑HER3 mAb. ADCC inducement
by HER3 was assayed as follows. Four female five‑week‑old
BALB/c nude mice (mean weight, 15±3 g) were purchased

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ASANO et al: ANTITUMOR ACTIVITIES OF AN ANTI-HER3 mAb

Figure 1. Production of anti‑HER3 mAbs. Procedure of the Cell‑Based Immunization and Screening (CBIS) method. (A) CHO/PA16‑HER3 cells were
immunized into BALB/c mice using an intraperitoneal injection. (B) Spleen cells were fused with P3U1 cells. (C) Culture supernatants were screened using
flow cytometry. (D) After limiting dilution and several additional screenings, an anti‑HER3 mAb was finally established. mAb, monoclonal antibody.

from Charles River Laboratories, Inc. Mice were kept under
specific pathogen‑free condition on an 11‑h light/13‑h darkcycle at a temperature of 23±2˚C and 55±5% humidity withfood and water supplied ad libitum during the experimental
periods. After euthanasia by cervical dislocation, spleens were
removed aseptically, and single‑cell suspensions were obtainedby forcing spleen tissues through a sterile cell strainer (productno. 352360; Corning, Inc.) with a syringe. Erythrocytes werelysed with a 10‑sec exposure to ice‑cold distilled water. The
splenocytes were washed with DMEM and resuspended in
DMEM with 10% FBS; this preparation was designated as
effector cells. The target tumor cells were labeled with 10 µg/ml
Calcein‑AM (Thermo Fisher Scientific, Inc.) and resuspended
in the same medium. The target cells were then transferred to
96‑well plates, at 2x104 cells/well, and mixed with effector cells
at an effector‑to‑target ratio of 100:1, along with 100 µg/ml ofanti‑HER3 antibodies or control mouse IgG2a. After a 4.5‑hincubation at 37˚C, Calcein release into the supernatant wasmeasured for each well. Fluorescence intensity was assessedusing a microplate reader (Power Scan HT; BioTek Instruments,Inc.) with an excitation wavelength of 485 nm and an emis‑
sion wavelength of 538 nm. Cytolytic activity was measured
as a percentage of lysis and calculated using the equation:
Percentage of lysis (%) = (E‑S)/(M‑S) x100, where E is the fluo‑
rescence measured in combined cultures of target and effector
cells, S is the spontaneous fluorescence of the target cells, andM is the maximum fluorescence measured after lysis of all cellswith buffer containing 0.5% Triton X‑100, 10 mM Tris‑HCl(pH 7.4), and 10 mM EDTA. Animal studies for ADCC wereapproved by the Institutional Committee for experiments of theInstitute of Microbial Chemistry (permit no. 2020‑024).
CDC activity of an anti‑HER3 mAb. CDC inducement by HER3
was assayed as follows. Target cells were labeled with 10 µg/ml
Calcein‑AM (Thermo Fisher Scientific, Inc.), resuspended
in medium and plated in 96‑well plates, at 2x10 4 cells/well,with 15% rabbit complement (Low‑Tox‑M rabbit complement;
Cedarlane Laboratories), 100 µg/ml of anti‑HER3 antibodies,or control IgG (mouse IgG2a) added to each well. After 4.5 hof incubation at 37˚C, Calcein release into the supernatant wasmeasured for each well. Fluorescence intensity was calculated
as described in the ADCC section above.
Antitumor activity of an anti‑HER3 mAb in xenografts of coloncancers. Sixteen five‑week‑old female BALB/c nude mice (meanweight, 15±3 g) were purchased from Charles River Laboratories,
Inc. All animal experiments were performed in accordancewith institutional guidelines and regulations to minimize
animal suffering and distress in the laboratory. The Institutional
Committee for experiments of the Institute of Microbial
Chemistry (permit no. 2020‑024) approved the animal studies
for antitumor activity here described. Mice were maintained in apathogen‑free environment on an 11‑h light/13‑h dark cycle at atemperature of 23±2˚C and 55±5% humidity with food and watersupplied ad libitum throughout the experiments. Mice were moni‑
tored for health and weight every three or five days. Experimentson mice were conducted in four weeks. Weight loss exceeding
25% or tumor volume exceeding 3,000 mm3 were identified as
humane endpoints for euthanasia. At humane and experimental
endpoints, mice were euthanized by cervical dislocation, anddeath was verified by validating respiratory and cardiac arrest.
After a one‑week acclimation period, these mice were usedin experiments at six weeks of age (mean weight, 16±2 g).
Caco‑2 cells (0.3 ml of 1.33x108 cells/ml in DMEM) weremixed with 0.5 ml BD Matrigel Matrix Growth Factor Reduced(BD Biosciences), and 100 µl of this suspension (5x106 cells)
was injected subcutaneously into the left flank of each animal.
On the eighth day post‑inoculation, 16 mice were divided into
two groups (n=8 in each group) with equal mean tumor volume:
An anti‑HER3 mAb group or a control mouse IgG group.
Then, 100 µg of an anti‑HER3 mAb or control mouse IgG in
100 µl PBS was injected i.p. Additional antibody inoculations
were performed on days 15 and 23. Twenty‑six days after cell
implantation, all mice were euthanized by cervical disloca‑
tion, and tumor diameters and volumes were measured andrecorded.

ONCOLOGY REPORTS 46: 173, 2021

5

Figure 2. Characterization of H3Mab‑17. (A) Confirmation of HER3 expression by western blot analysis. Cell lysates were electrophoresed and transferred onto
PVDF membranes. After blocking, PVDF membranes were incubated with an anti‑HER3 mAb (clone D22C5) or anti‑β‑actin (clone AC‑15), followed by incuba‑tion with peroxidase‑conjugated anti‑rabbit immunoglobulins or peroxidase‑conjugated anti‑mouse immunoglobulins. Blots were developed using ImmunoStar
LD or ECL Plus Western Blotting Substrate and imaged with a Sayaca‑Imager. (B) Flow cytometry analysis. CHO‑K1, CHO/HER3‑PA, MCF7, and BINDS‑30
cells were treated with 1 µg/ml of H3Mab‑17, followed by treatment with Alexa Fluor 488‑conjugated anti‑mouse IgG. Black line, negative control. (C) Flow cytom‑
etry analysis. Colon cancer cell lines, such as Caco‑2, HCT 116, HCT‑15, HT‑29, LS 174T, COLO 201, COLO 205, HCT‑8, SW1116, and DLD‑1 cells were treatedwith 1 µg/ml of H3Mab‑17, followed by treatment with Alexa Fluor 488‑conjugated anti‑mouse IgG. Black line, negative control. mAb, monoclonal antibody.

Statistical analyses. All data are expressed as mean ± standard
error of the mean (SEM). Statistical analysis was conducted withANOVA and Tukey's multiple comparisons tests for ADCC and
CDC, ANOVA and Sidak's multiple comparisons tests for tumor
volume and mouse weight, and Welch's t‑test for tumor weight.
All calculations were performed with GraphPad Prism 8(GraphPad Software, Inc.). A P‑value of <0.05 was considered
to indicate a statistically significant difference.
Results
Development of anti‑HER3 mAbs. We employed the CBIS
method to develop anti‑HER3 mAbs using CHO/PA16‑HER3

cells both for the immunization and flow cytometry screening(Fig. 1). The developed hybridomas were seeded into 96‑wellplates and cultivated for 10 days. Supernatants positive for
CHO/HER3‑PA and negative for CHO‑K1 were selectedby flow cytometry. After limiting dilution and several addi‑
tional screenings, an anti‑HER3 mAb, H3Mab‑17 (mouse
IgG2a, kappa), was finally established.
Confirmation of HER3 expression by western blot analysis.
We established CHO/mock (Ble), CHO/PA16‑HER3,
CHO/mock (Neo), and CHO/HER3‑PA, and investigatedwhether HER3 was overexpressed in those cell lines. As shownin Fig. 2A, overexpression of HER3 in CHO/PA16‑HER3 and6

ASANO et al: ANTITUMOR ACTIVITIES OF AN ANTI-HER3 mAb

Figure 3. Determination of the binding affinity of H3Mab‑17. CHO/HER3‑PA
and Caco‑2 were suspended in 100 µl of serially diluted H 3Mab‑17(0.006‑25 µg/ml) prior to Alexa Fluor 488‑conjugated anti‑mouse IgG
addition. Fluorescence data were collected using a BD FACSLyric. K D,
dissociation constant.

CHO/HER3‑PA was confirmed by western blot analysis using
an anti‑HER3 mAb (clone D22C5). Endogenous HER3 expres‑sion in MCF7 cells was also detected by an anti‑HER3 mAb.
In contrast, knockout of endogenous HER3 in BINDS‑30(MCF7/HER3‑KO) was confirmed by western blot analysis
using an anti‑HER3 mAb.
Flow cytometry analyses of H3Mab‑17. We performed flow
cytometry using H3Mab‑17 against CHO‑K1, CHO/HER3‑PA,
MCF7, and BINDS‑30 (MCF7/HER3‑KO). H3Mab‑17 recog‑
nized the CHO/HER3‑PA cells, but not the parental CHO‑K1
cells (Fig. 2B). H3Mab‑17 also recognized the endogenous
HER3 in MCF7 breast cancer cells (Fig. 2B). The reaction of
H3Mab‑17 to BINDS‑30 was lost after the knockout of HER3
in MCF7 cells (Fig. 2B), indicating the specificity of H3Mab‑17
for HER3.
Next, we investigated whether H3Mab‑17 reacts with coloncancer cell lines. As shown in Fig. 2C, H3Mab‑17 reacted with10 colon cancer cell lines, Caco‑2, HCT 116, HCT‑15, HT‑29,
LS 174T, COLO 201, COLO 205, HCT‑8, SW1116, and DLD‑1.
Among them, Caco‑2 was known to be useful for the mouse
xenograft model (55). Therefore, we used Caco‑2 cells for the
ADCC/CDC assay or in vivo xenograft models.
Determination of the binding affinity of H3Mab‑17. A kinetic
analysis of the interactions of H3Mab‑17 with CHO/HER3‑PA
and Caco‑2 cells was then conducted using flow cytometry.
The K D for H3Mab‑17 in CHO/HER3‑PA and Caco‑2 cells

Figure 4. Evaluation of ADCC and CDC activities by H3Mab‑17. (A) ADCC
activities by H3Mab‑17, control mouse IgG2a, and control PBS in Caco‑2cells. (B) CDC activities by H 3Mab‑17, control mouse IgG2a, and control
PBS in Caco‑2 cells. Values are mean ± SEM. Asterisk indicates statisticalsignificance (*P<0.05; n.s., not significant, ANOVA and Tukey‑Kramer's test).
ADCC, antibody‑dependent cellular cytotoxicity; CDC, complement‑depen‑
dent cytotoxicity.

were 3.0x10‑9 and 1.5x10‑9 M, respectively (Fig. 3), indicatinghigh binding affinity of H3Mab‑17 against HER3‑expressing
cells.
ADCC and CDC activities of H3Mab‑17 in colon cancer celllines. We then examined whether H3Mab‑17 (mouse IgG2a)
induced ADCC and CDC activity in HER3‑expressing Caco‑2colon cancer cell lines. H3Mab‑17 exhibited higher ADCC
(14.8% cytotoxicity) in Caco‑2 cells than that of control mouse
IgG2a (5.2% cytotoxicity; P<0.05) or control PBS (3.2% cyto‑
toxicity; P<0.05) treatment (Fig. 4A). H 3Mab‑17 was also
associated with a more robust CDC activity (30.4% cyto‑
toxicity) in Caco‑2 cells than the control mouse IgG2a(7.7% cytotoxicity; P<0.05) or the control with PBS treat‑ment (8.8% cytotoxicity; P<0.05) (Fig. 4B). These favorable
ADCC/CDC activities indicated that H3Mab‑17 may induce
strong antitumor activity against colon cancer cells in vivo aswell as in vitro.
Antitumor effect of H3Mab‑17 in mouse xenografts of coloncancer cells. Tumor formation of 16 Caco‑2‑bearing mice wasobserved on day eight. Then, these 16 Caco‑2‑bearing mice

ONCOLOGY REPORTS 46: 173, 2021

Figure 5. Evaluation of antitumor activity of H3Mab‑17 in Caco‑2 xenografts.
(A) Caco‑2 cells (5x106 cells) were injected subcutaneously into the left flank.
After day 8, 100 µg of H3Mab‑17 and control mouse IgG in 100 µl PBS were
injected i.p. into the treated and control mice, respectively. Additional anti‑
bodies were then injected on days 15 and 23. Tumor volume was measured
on days 8, 11, 15, 18, 23 and 26. Values are mean ± SEM. Asterisk indicatesstatistical significance (**P<0.01; n.s., not significant, ANOVA and Sidak's
multiple comparisons test). , control; r, H3Mab‑17. (B) Tumors of Caco‑2
xenografts were resected from H3Mab‑17 and control mouse IgG groups.
Tumor weight on day 26 was measured from excised xenografts. Values aremean ± SEM. Asterisk indicates statistical significance (**P<0.01, Welch's
t‑test). , control; r, H3Mab‑17. (C) Resected tumors of Caco‑2 xenografts
from H3Mab‑17 and control mouse IgG groups on day 26. The tumor in thesquare dotted region was the largest tumor in this experiment. The verticaland horizontal lengths for Caco‑2 cells were 1.6 and 1.3 cm, respectively(estimated tumor volume, 1,352 mm3, tumor weight, 661 mg). Scale bar, 1 cm.

were divided into an H3Mab‑17‑treated group and a control
group. On days 8, 15 and 23 after Caco‑2 cell injections into
the mice, H3Mab‑17 (100 µg) or control mouse IgG (100 µg)
were injected i.p. in the Caco‑2 xenograft model mice. The
tumor volume was measured on days 8, 11, 15, 18, 23 and 26
after the Caco‑2 cell injection. H3Mab‑17‑treated mice exhib‑
ited significantly less tumor growth on day 18 (P<0.01), day 23(P<0.01), and day 26 (P<0.01), compared with the IgG‑treatedcontrol mice (Fig. 5A). The reduction in the volume of thetumors by H3Mab‑17 treatment was of 54% on day 26. Tumors
from the H3Mab‑17‑treated mice weighed significantly lessthan tumors from the IgG‑treated control mice (59% reduc‑tion, P<0.01; Fig. 5B). Resected tumors on day 26 are presented

7

Figure 6. Body weights of mice implanted with Caco‑2 xenografts. Caco‑2cells (5x10 6 cells) were injected subcutaneously into the left flank. After
day 8, 100 µg of H 3Mab‑17 and control mouse IgG in 100 µl PBS were
injected i.p. into treated and control mice, respectively. Additional antibodies
were then injected on days 15 and 23. (A) Body weights of the mice with theCaco‑2 xenografts of H3Mab‑17 group and control group were recorded ondays 8, 11, 15, 18, 23 and 26. Values are mean ± SEM. n.s. indicates no statis‑
tical significance (ANOVA and Sidak's multiple comparisons test). (B) Body
appearance of mice with the Caco‑2 xenografts of H 3Mab‑17 and control
mouse IgG groups on day 26. Scale bar, 1 cm.

in Fig. 5C. The total body weights did not significantly differ
between the treatment and control groups (Fig. 6A and B).
These results indicated that H3Mab‑17 reduced the growth of
Caco‑2 xenografts, without eliminating them completely.
Discussion
Many commercially available anti‑HER3 mAbs have beendeveloped using recombinant HER3 protein, peptide or
cDNA as an immunogen. Seribantumab was developed byphage display (56,57) and lumretuzumab was developed
using recombinant HER3 extracellular domain as an immu‑
nogen (46). In this study, we succeeded in the developmentof an anti‑HER3 mAb using the CBIS method, which used
HER3‑expressed cells for both immunization and screening.
The CBIS method can help us effectively develop mAbs
that are useful in flow cytometry. We recently succeeded indeveloping numerous useful mAbs that target membrane

8

ASANO et al: ANTITUMOR ACTIVITIES OF AN ANTI-HER3 mAb

proteins, including podoplanin (58‑61), CD20 (62), CD44 (63),CD133 (54), and TROP2 (64,65). Importantly, these mAbs arevery useful for various experiments, including not only flow
cytometry, but also western blot analysis and immunohisto‑
chemistry. Furthermore, those mAbs possess ADCC/CDC
activities and antitumor activities (61). Using the CBIS method,
proteins for immunogen expressed on cells maintain its
native conformation and glycosylation pattern. Previously,
we successfully established a cancer‑specific mAb (CasMab)against podoplanin, which recognizes the cancer cell‑specificglycosylation of podoplanin (66). Therefore, we may develop
CasMab against HER3 using the CBIS method in the future.
The CBIS method is advantageous for the development for
specific and sensitive mAbs for antibody therapy.
New highly accurate therapeutic options are possible totreat most solid tumors. In the case of colorectal cancer, HER3
overexpression is found in ~17‑75%, although the definition of
its cutoff signals for HER3 expression are different in each
immunohistochemical study (67). It has been reported that theincidence of HER3 overexpression in metastatic colorectal
cancer is much higher than that of HER2 (68). In thisstudy, we developed an anti‑HER3 mAb, H3Mab‑17, which
specifically reacted with endogenous HER3 in colorectal
carcinoma cell lines in flow cytometry. The KD for H3Mab‑17
in CHO/HER3‑PA and Caco‑2 cells were determined to be
3.0x10‑9 and 1.5x10‑9 M, respectively, suggesting high binding
affinity of H3Mab‑17 for HER3. In vitro experiments revealed
strong ADCC and CDC inducement against Caco‑2 cells by
H3Mab‑17. In vivo experiments on Caco‑2 xenografts revealed
that the treatment with H3Mab‑17 significantly reduced thetumor growth, compared with the control mouse IgG. Basedon these findings, H3Mab‑17 may be useful in therapeutic
approach for patients with colorectal cancer.
Although H 3Mab‑17 recognizes both overexpressedand endogenous HER3 by flow cytometric analyses, it is
not applicable to western blot and immunohistochemical
analyses (data not shown). H3Mab‑17 did not recognize dena‑
tured HER3, such as SDS‑treated and formalin‑fixed HER3
probably because it might recognize the three‑dimensional
structure of HER3. Since the antitumor activity mechanismof H3Mab‑17 has not been clarified, we need to identify the
epitope of H3Mab‑17 and investigate the inhibitory activityof HER3‑neureglin interaction of H3Mab‑17. Furthermore,
HER3‑ADC and HER3‑chimeric antigen receptor (CAR)‑T
should be developed in future research.
Acknowledgements
We would like to thank Ms. Miyuki Yanaka, Ms. Saori Handa,
and Mr. Yu Komatsu (Department of Antibody Drug
Development, Tohoku University Graduate School of
Medicine) for technical assistance in the in vitro experiments,and Ms. Akiko Harakawa [Institute of Microbial Chemistry(BIKAKEN), Numazu, Microbial Chemistry Research
Foundation] for technical assistance in the animal experiments.
Funding
This research was supported in part by the Japan Agency
for Medical Research and Development (AMED) under

grant nos. JP21am0401013 (to YK) and JP21am0101078 (toYK), and by the Japan Society for the Promotion of Science(JSPS) Grants‑in‑Aid for Scientific Research (KAKENHI)
grant nos. 21K15523 (to TA), 21K07168 (to MKK), 19K07705(to YK) and 20K16322 (to MS).
Availability of data and materials
The datasets used and/or analyzed during the study are avail‑able from the corresponding author on reasonable request.
Authors' contributions
TA, TO, TN, RN, HHo, TT, and MS performed the experi‑ments. JT and MKK analyzed the experimental data. HHa,
MK, and YK designed the present study. TA, TO, and YK
wrote the manuscript. All the authors read and approved thefinal manuscript for publishing.
Ethics approval and consent to participate
The Animal Care and Use Committee of Tohoku University
approved all the animal experiments (permit no. 2019NiA‑001).
Animal studies for ADCC and the antitumor activity wereapproved by the Institutional Committee for experiments of
the Institute of Microbial Chemistry (permit no. 2020‑024).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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