MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 4, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0017

Anti-Human Epidermal Growth Factor Receptor 2Monoclonal Antibody H2Mab-41 Exerts Antitumor Activity
in a Mouse Xenograft Model of Colon Cancer

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Yukinari Kato,1,2 Tomokazu Ohishi,3 Shinji Yamada,1 Shunsuke Itai,1,4 Junko Takei,1,4 Masato Sano,1Takuro Nakamura,1 Hiroyuki Harada,4 Manabu Kawada,3 and Mika K. Kaneko1

The expression of human epidermal growth factor receptor 2 (HER2) has been reported to be overexpressed inseveral cancers, such as breast, lung, gastric, pancreatic, and colorectal cancers, and be associated with poor
clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits forpatients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel antiHER2 monoclonal antibody (mAb), H2Mab-41 (IgG2b, kappa), and the antitumor activity of H2Mab-41 wasinvestigated using mouse xenograft models. Caco-2 cells (human colon cancer cell line), which expresses
HER2, were subcutaneously implanted into the flanks of nude mice. H2Mab-41 and control mouse IgG wereinjected three times into the peritoneal cavity of mice. H2Mab-41 significantly reduced tumor development of
Caco-2 xenograft in comparison with the control mouse IgG on days 5, 8, 11, 15, and 19. Taken together, theseresults suggest that H2Mab-41 is useful for antibody therapy against HER2-expressing colon cancers.
Keywords: HER2, monoclonal antibody, antitumor activity, colon cancer
Introduction

Trastuzumab and pertuzumab, humanized anti-humanepidermal growth factor receptor 2 (HER2) monoclonal
antibodies (mAbs), and trastuzumab emtansine, an antibody–
drug conjugate (ADC), have been approved for the treatment of
HER2-positive breast cancer.(1–3) Trastuzumab treatment
has resulted in significant survival benefits for patients withmetastatic HER2-positive breast cancer,(4) although HER2
is associated with poor clinical outcomes.(5,6) Furthermore,
the combination of pertuzumab and trastuzumab with chemotherapy has led to significant improvements in overall
survival compared with trastuzumab alone plus chemotherapy.(7) HER2 overexpression has been also reported in gastriccancers,(8) pancreatic cancers,(9) lung cancers,(10) and colorectal cancers.(11) In this study, we developed a novel anti-HER2
mAb H2Mab-41, and investigated its antitumor activity againstmouse xenograft models of human colon cancer.
Materials and Methods
Cell lines

P3U1, LN229, and Caco-2 were obtained from the American
Type Culture Collection (ATCC, Manassas, VA). LN229/HER2

cells were established in our previous study.(12) P3U1 was cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto,Japan), and LN229, LN229/HER2, and Caco-2 were cultured
in the Dulbecco’s modified Eagle’s medium (DMEM) medium (Nacalai Tesque, Inc.) supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific,Inc., Waltham, MA), 100 U/mL of penicillin, 100 mg/mL ofstreptomycin, and 25 mg/mL of amphotericin B (NacalaiTesque, Inc.) at 37C in a humidified atmosphere containing
5% CO2 and 95% air.
Hybridoma production

Female 4-week-old BALB/c mice were purchased fromCLEA Japan (Tokyo, Japan). Animals were housed underspecific pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved all the animal
experiments described in this study. Anti-HER2 hybridomas
were produced, as described previously.(12) In brief, BALB/cmice were immunized using intraperitoneal (i.p.) injectionsof 100 mg of recombinant HER2-extracellular domain together with Imject Alum (Thermo Fisher Scientific, Inc.).
After several additional immunizations, a booster injection
was intraperitoneally administered 2 days before harvesting

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
3
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Japan.
4
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,Tokyo, Japan.
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spleen cells. Spleen cells were then fused with P3U1 cells
using PEG1500 (Roche Diagnostics, Indianapolis, IN). The
resulting hybridomas were grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.).
Culture supernatants were screened using enzyme-linked
immunosorbent assay with recombinant HER2-extracellulardomain. mAbs were purified from the supernatants of hybridomas, cultured in Hybridoma-SFM medium (ThermoFisher Scientific, Inc.) using Protein G Sepharose 4 Fast Flow(GE Healthcare UK Ltd., Buckinghamshire, England).

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Flow cytometry

Cells were harvested by brief exposure to 0.25% trypsin/1-mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.).
After washing with 0.1% bovine serum albumin/phosphatebuffered saline (PBS), the cells were treated with 1 mg/mL ofanti-HER2 (H2Mab-41 and H2Mab-139) for 30 minutes at 4Cand subsequently with Alexa Fluor 488-conjugated anti-mouse
IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA).

KATO ET AL.

Fluorescence data were collected using EC800 Cell Analyzers(Sony Corp., Tokyo, Japan).
Antitumor activity of H2Mab-41

Female BALB/c nude mice (6-week old) were purchasedfrom Charles River (Kanagawa, Japan) and used in experiments when they were 10 weeks old. Caco-2 (0.3 mL of
1.33 · 108/mL in DMEM) were mixed with 0.5 mL of BD
Matrigel Matrix Growth Factor Reduced (BD Biosciences,San Jose, CA). A 100-mL suspension (containing 5 · 106cells) was injected subcutaneously into the left flanks of nude
mice. After day 1, 100 mg of H2Mab-41 and control mouse
IgG (Sigma-Aldrich Corp., St. Louis, MO) in 100 mL PBS
were injected into the peritoneal cavity of each mouse. Additional antibodies were then injected on days 8 and 15. The
tumor diameter and volume were determined as previously
described.(13) The mice were euthanized 19 days after cell
implantation. All data were expressed as mean – SEM. Statistical analysis was performed using the Tukey–Kramer test.
p < 0.05 was considered to be statistically significant.

FIG. 1. Flow cytometric analysis of H2Mab-41 against colon cancer cell lines. Cells were treated with 1 mg/mL of H2Mab41 (A) and H2Mab-139 (B), followed by Alexa Fluor 488-conjugated anti-mouse IgG; left line, negative control.

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ANTI-HER2 MAB AGAINST COLON CANCER

FIG. 2. Evaluation of antitumor activity (tumor volume) ofH2Mab-41 against Caco-2 xenograft model. Tumor volumeof Caco-2 xenografts. Caco-2 cells were injected subcutaneously into female nude mice. The indicated antibodies(100 mg/day; 5 mg/kg) were administered intraperitoneallyon days 1, 8, and 15 after cell inoculation. The tumor volume
was measured at the indicated time points. The values arepresented as mean – SEM. **p < 0.01, Tukey–Kramer’s test.

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ded compared with LN229 because LN229 cells endogenously express HER2. We obtained one clone H2Mab-41 of
IgG2a subclass although almost all mAbs were determined tobe a mouse IgG1 subclass.
We first characterized H2Mab-41 in flow cytometry.
H2Mab-41 reacted with LN229/HER2 cells, weakly reactedwith LN299 cells (Fig. 1A), indicating that H2Mab-41 is
specific for HER2. As a positive control, H2Mab-139 also
reacted in the same pattern (Fig. 1B). Furthermore, H2Mab41 recognized endogenous HER2 in 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 (Fig. 1A) inthe same pattern with H2Mab-139 (Fig. 1B).
To study the antitumor activity of H2Mab-41 on cell
growth in vivo, Caco-2 cells were subcutaneously implanted
into the flanks of nude mice. H2Mab-41 and control mouse
IgG were injected three times (on days 1, 8, and 15 after cellinjections) into the peritoneal cavity of mice. Tumor formation was observed in mice from the control and H2Mab-41treated groups in Caco-2 xenograft models. H2Mab-41
significantly reduced the tumor development of Caco-2 xenograft in comparison with that in control mouse IgG on days
5, 8, 11, 15, and 19 (Fig. 2). The resected tumors of Caco-2
xenografts are depicted in Figure 3A. The tumor weight ofResults

In this study, we immunized one mouse with the recombinant extracellular domain of HER2,(14) which was purified
using MAP tag system.(15) Flow cytometry was performed tocheck reactions with LN229 (a glioblastoma cell line) andHER2-overexpressing LN229 (LN229/HER2) cells (data notshown). A stronger reaction against LN229/HER2 was nee-FIG. 3. Evaluation of antitumor activity (tumor weight) of
H2Mab-41 against Caco-2 xenograft model. (A) Resected
tumors of Caco-2 xenografts. (B) Tumor weight of Caco-2
xenografts (day 19). *p < 0.05, Tukey–Kramer’s test.

FIG. 4. Evaluation of antitumor activity (body weight) ofH2Mab-41 against Caco-2 xenograft model. (A) Caco-2xenograft mice models on day 19. (B) Body weight of Caco2 xenografts (day 19). The values are presented as mean –SEM. n.s., not significant.

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mice in H2Mab-41-treated group was significantly lower thanthat in the control mouse IgG group in Caco-2 xenograftmodels (Fig. 3B). Caco-2 xenograft mice models on day 19
are shown in Figure 4A. Body weight was not significantly
different among the two groups in the Caco-2 xenograftmodels (Fig. 4B).

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Discussion

Recently, trastuzumab deruxtecan (also known as DS-8201)
was reported to be an ADC, which comprised trastuzumab, anovel enzyme-cleavable linker, and a topoisomerase I inhibitor
payload.(16) Of interest, trastuzumab deruxtecan showed antitumor activity, even in low HER2-expressing tumors. Trastuzumab deruxtecan has several innovative features: (i) a highly
potent novel payload with a high drug-to-antibody ratio, (ii)good homogeneity, (iii) a tumor-selective cleavable linker, (iv)a stable linker-payload in circulation, and (v) a short systemic
half-life cytotoxic agent in vivo.(17) Furthermore, the released
cytotoxic payload could exert a bystander effect.(17) Although
ADC technology is very critical technology, the developmentof more functional mAbs might be still necessary.
Recently, we developed several anti-HER2 mAbs, such as
H2Mab-77,(12) H2Mab-119,(18) and H2Mab-139(14) using CasMab technology.(19) Those anti-HER2 mAbs are useful for flow
cytometry, Western blot, and immunohistochemical analyses.
However, those three mAbs do not possess antibody-dependent
cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) because the subclass of those mAbs is mouse
IgG1. Among the mouse IgG subclasses, IgG2a(20) and IgG2b(21)are known to possess ADCC and CDC. Then, we successfully
screened H2Mab-41 of IgG2b subclass. Unfortunately, H2Mab41 did not react with HER2 in Western blot and immunohistochemical analyses (data not shown). In contrast, H2Mab-41
exerted antitumor activity against a colon cancer xenograft,indicating that H2Mab-41 is applicable for antibody therapy
against human colon cancers expressing HER2. Further studies
on antitumor activities against HER2-expressing xenografts
are, therefore, necessary to obtain a more detailed understanding of antibody therapy against HER2.
Acknowledgments

We thank Akiko Harakawa, Miyuki Yanaka, Kayo Hisamatsu, Saori Handa, and Yoshimi Nakamura for their excellent technical assistance. This research was supported inpart by AMED under Grant Nos. JP18am0101078 (Y.K.),
JP18am0301010 (Y.K.), and JP18ae0101028 (Y.K.), and byJSPS KAKENHI Grant Nos. 17K07299 (M.K.K.) and16K10748 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.
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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
MiyagiJapanE-mail: yukinarikato@med.tohoku.ac.jp;
yukinari-k@bea.hi-ho.ne.jp
Received: March 8, 2019
Accepted: May 17, 2019