MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 4, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0013

An Antihuman Epidermal Growth Factor Receptor 2
Monoclonal Antibody (H2Mab-19) Exerts Antitumor Activity
in Glioblastoma Xenograft Models

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Yukinari Kato,1,2 Tomokazu Ohishi,3 Junko Takei,1 Takuro Nakamura,1Manabu Kawada,3 and Mika K. Kaneko1

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in glioblastoma as wellas breast, gastric, lung, colorectal, and pancreatic cancers. Its expression is associated with poor clinical outcomes. Anti-HER2 antibodies have provided significant survival benefits to patients with HER2-overexpressing
breast and gastric cancers. We recently developed an anti-HER2 monoclonal antibody (mAb), H2Mab-19(IgG2b, kappa), by immunizing mice with the extracellular domain of HER2, which is expressed in LN229
glioblastoma cells. In this study, we investigated the antitumor activity of H2Mab-19 in an LN229 glioblastoma
xenograft model. H2Mab-19 showed high binding affinity (KD: 1.1 · 10-8 M) against LN229 cells. Furthermore,
H2Mab-19 significantly reduced tumor development in an LN229 xenograft. These results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing glioblastomas.
Keywords: HER2, monoclonal antibody, antitumor activity, glioblastoma
Introduction

Gliomas represent the most common primary brain
tumor, and glioblastoma multiforme is the most frequent and malignant type of glioma.(1) Despite advances insurgical techniques, radiation therapy, and adjuvant chemotherapy, their prognoses remain poor. Many antigens, including epidermal growth factor receptor (EGFRwt), its
glioma-associated deletion variant EGFRvIII, tenascin, chondroitin sulfate proteoglycans, and lacto-series gangliosides,
have been found in gliomas, and upregulation of those molecules has been observed in brain tumor cells.(2) Althoughthese molecules are under investigation as therapeutic targets,
multiple entities may ultimately have to be targeted to compensate for tumor heterogeneity. Human epidermal growth
factor receptor 2 (HER2) has also been reported to be expressed in glioblastomas, and represents one of the moleculartargets for immunotherapy.(3–8)
A novel anti-HER2 monoclonal antibody (mAb) (H2Mab19; IgG2b, kappa) was recently developed by immunizing micewith the purified recombinant ectodomain of HER2.(9) H2Mab19 exerts antitumor activity in BT-474, HSC-2, and SAS xenografts by antibody-dependent cellular cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC), suggestingthat treatment with H2Mab-19 may be a useful therapy forpatients with HER2-expressing breast and oral cancers. In this1
2
3

study, we investigated whether H2Mab-19 exhibits antitumor
activity in a mouse xenograft model of glioblastoma.
Materials and Methods
Cell lines

LN229 (glioblastoma cell line) was obtained from theAmerican Type Culture Collection (Manassas, VA). LN229/
HER2 was established in a previous study.(10) LN229 and
LN229/HER2 were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) (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, and 0.25 lg/mL ofamphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified
atmosphere containing 5% CO2 and 95% air.
Flow cytometry

LN229 or LN229/HER2 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 in phosphate-buffered saline (PBS), LN229 or
LN229/HER2 cells were treated with 1 lg/mL anti-HER2(H2Mab-19) for 30 minutes at 4C and subsequently with

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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136

Alexa Fluor 488-conjugated antimouse IgG (1:1000; Cell
Signaling Technology, Inc., Danvers, MA). Fluorescencedata were collected using an EC800 Cell Analyzer (SonyCorp., Tokyo, Japan).
Determination of the binding affinity

LN229 cells were suspended in 100 lL serially diluted
H2Mab-19 (6 ng/mL–100 lg/mL), followed by the additionof Alexa Fluor 488-conjugated antimouse IgG (1:200; Cell
Signaling Technology, Inc.). Fluorescence data were collected using an EC800 Cell Analyzer (Sony Corp.). The
dissociation constant (KD) was obtained by fitting binding
isotherms to built-in one-site binding models in GraphPad
PRISM 6 (GraphPad Software, Inc., La Jolla, CA).

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Antitumor activity of H2Mab-19 in the xenograftsof glioblastoma

Sixteen 6-week-old female BALB/c nude mice were purchased from Charles River Laboratories and used at 7 weeks

KATO ET AL.

of age. All animal experiments were performed in accordance with relevant guidelines and regulations to minimize
animal suffering and distress in the laboratory. Animal studies for antitumor activity were approved by the institutional
committee for experiments of the Institute of Microbial
Chemistry (Permit number: 2019-021). Mice were monitored
for health and weight every 2 to 4 days. The duration of theexperiments was 3 weeks. A bodyweight loss exceeding 25%
and a maximum tumor size exceeding 3000 mm3 were designated as humane endpoints. Mice were euthanized by cervical dislocation and death was verified by respiratory arrest
and cardiac arrest.
LN229 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). One hundred
microliters of this suspension (5 · 106 cells) was injectedsubcutaneously into the left flank. After day 1, 100 lg of
H2Mab-19 and control mouse IgG (Sigma-Aldrich Corp.) in
100 lL of PBS were injected intraperitoneal (i.p.) into treatedand control mice, respectively. Additional antibodies were

FIG. 1. Characterization of H2Mab-19 using flow cytometry. (A) LN229 cells were treated with H2Mab-19. The blackline represents the negative control (blocking buffer). (B) Determination of the binding affinity of H2Mab-19 for LN229
cells using flow cytometry. H2Mab-19, anti-human epidermal growth factor receptor 2 monoclonal antibody.

AN ANTI-HER2 MAB FOR GLIOBLASTOMAS

then injected on days 6 and 14. Twenty days after cell implantation, all mice were euthanized by cervical dislocationand tumor diameters and volumes were determined as previously described.(11)
Statistical analyses

Results
Characterization of H2Mab-19 against a glioblastoma
cell line

We first measured the surface expression of HER2 inthe LN229 human glioblastoma and HER2-overexpressing
LN229/HER2 cell lines. As expected, H2Mab-19 recognizedendogenous HER2 in LN229 cells and overexpressed HER2of LN229/HER2 cells by flow cytometry (Fig. 1A).
The binding affinity (KD) of H2Mab-19 to LN229 cells was
1.1 · 10-8 M (Fig. 1B), indicating that H2Mab-19 exhibits a

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All data are expressed as mean – standard error of themean. Statistical analysis used analysis of variance andTukey–Kramer’s test with R statistical. p values < 0.05 wereconsidered statistically significant.

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FIG. 2. Evaluation of the antitumor activity of H2Mab-19. (A) Tumor volume was measured in LN229 xenografts.
(B) Resected tumors of LN229 xenografts (day 20). (C) Tumor weight was measured in LN229 xenografts (day 20).
(D) Appearance of treated mice on day 20. (E) Body weights of the mice with the LN229 xenografts were measured for
20 days. Values represent the mean – SEM. **p < 0.01, *p < 0.05, the Tukey–Kramer’s test. n.s.: not significant. SEM,
standard error of the mean.

138high affinity for HER2-expressing glioblastoma cell lines.
These results suggest the possibility of targeting HER2 as an
antigen for immunotherapy.

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Antitumor activity of H2Mab-19 in mouse xenograftsof glioblastoma

Next, we investigated whether H2Mab-19 induces cytotoxic activity against glioblastoma in vivo. To study the antitumor activity of H2Mab-19 on cell growth in vivo, LN229
cells were implanted subcutaneously in the flanks of nude
mice. H2Mab-19 and control mouse IgG were injected i.p.
three times (days 1, 6, and 14 after cell injection) into treatedand control mice, respectively. Tumor formation was observed in both the H2Mab-19-treated and control groups.
H2Mab-19 treatment significantly reduced tumor development compared with development in control mice on day
10 ( p < 0.05), day 14 ( p < 0.01), day 17 ( p < 0.01), and day
20 ( p < 0.01; Fig. 2A). Resected tumors are depicted in
Figure 2B. Tumor weight from H2Mab-19-treated mice wassignificantly less than that of the IgG-treated control mice
( p < 0.05; Fig. 2C). Mice on day 20 are depicted in Figure 2D.
Total body weight was not significantly different between thetwo groups (Fig. 2E). These results indicate that H2Mab-19
exerts a significant antitumor effect against HER2-expressing
glioblastomas.
Discussion

HER2 overexpression has been reported in breast andgastric cancers, and is associated with poor clinical outcomes.(12–14) The humanized anti-HER2 mAbs, trastuzumab
and pertuzumab, have been used in the treatment of HER2positive breast cancer.(15–17) Treatment with trastuzumab
resulted in significant survival benefits in these patients.(18)The combination of trastuzumab and pertuzumab with chemotherapy has led to significant improvements in overall
survival.(19) Recently, trastuzumab deruxtecan (DS-8201),
which consists of three components, a novel enzymecleavable linker, and a topoisomerase I inhibitor, exerts antitumor activity even in low-HER2–expressing tumors.(20,21)
Therefore, we still require additional sensitive and specificanti-HER2 mAbs, the epitope of which is different than
trastuzumab and pertuzumab. We have tried to develop manyanti-HER2 mAbs that react with not only breast cancers,(10)but also colon cancers,(22) pancreatic cancers,(23) and glioblastoma using CasMab technology.(24)
We previously developed anti-HER2 mAbs, H2Mab77,(10) H2Mab-119,(23) and H2Mab-139.(22) These antibodies
are useful for flow cytometry, Western blot, and immunohistochemical analyses. The subclass of these mAbs wasdetermined to be mouse IgG1. Therefore, they do not possess
ADCC or CDC activities. We further tried to develop an antiHER2 mAb of the IgG2b subclass using CasMab technology,
because mouse IgG2b antibodies exhibit ADCC and CDC
activity.(25) A novel anti-HER2 mAb (H2Mab-19) of IgG2b
was recently established,(9) which exhibited antitumor activity in breast or oral cancer xenografts by ADCC and
CDC. In this study, we investigated whether H2Mab-19
exhibits antitumor activities in LN229 mouse xenograftmodels, since HER2 has been reported to be expressed inglioblastomas.(3–8)

KATO ET AL.

Antigen–antibody affinity is thought to be an important factor
influencing the outcome of antibody-based therapy.(26) The
binding affinity (KD) of H2Mab-19 to LN229 was determined tobe 1.1 · 10-8 M using flow cytometry, indicating that H2Mab-19
shows high affinity to HER2-expressing glioblastoma cell lines.
This is similar to 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).(9)We selected an LN229 cell line for the in vivo study, since
LN229 is useful for glioblastoma xenograft models.(27) In ourprevious study, H2Mab-19 treatment significantly reduced
tumor development in breast and oral cancer xenograftmodels.(9) H2Mab-19 also exerted antitumor activity against
LN229 xenografts in this study (Fig. 2). Further studies using other glioblastoma xenografts should be performed toconfirm that H2Mab-19 could represent a viable therapy forpatients with HER2-expressing glioblastomas.
Acknowledgment

We thank Akiko Harakawa for technical assistance.
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 JP20am0401013 (Y.K.), JP20am0101078 (Y.K.),
and JP20ae0101028 (Y.K.), and by Japan Society for the
Promotion of Science ( JSPS) KAKENHI under Grant
Numbers 17K07299 (M.K.K.) and 19K07705 (Y.K.).
References

1. DeAngelis LM: Brain tumors. N Engl J Med 2001;344:
114–123.
2. Kato Y, Kuan CT, Chang J, Kaneko MK, Ayriss J, Piao H,
Chandramohan V, Pegram C, McLendon RE, Fredman P,
Mansson JE, and Bigner DD: GMab-1, a high-affinity anti3¢-isoLM1/3¢,6¢-isoLD1 IgG monoclonal antibody, raisedin lacto-series ganglioside-defective knockout mice. Biochem Biophys Res Commun 2010;391:750–755.
3. Shen L, Li H, Bin S, Li P, Chen J, Gu H, and Yuan W: The
efficacy of third generation antiHER2 chimeric antigen receptor T cells in combination with PD1 blockade against
malignant glioblastoma cells. Oncol Rep 2019;42:1549–1557.
4. Migliorini D, Dietrich PY, Stupp R, Linette GP, Posey AD,
Jr., and June CH: CAR T-cell therapies in glioblastoma: A
first look. Clin Cancer Res 2018;24:535–540.
5. Schneider JR, Kwan K, and Boockvar JA: Use of HER2specific chimeric antigen receptor-modified virus-specific
T cells as a potential therapeutic for progressive HER2positive glioblastoma. Neurosurgery 2017;81:N42–N43.
6. Ahmed N, Brawley V, Hegde M, Bielamowicz K, Kalra M,
Landi D, Robertson C, Gray TL, Diouf O, Wakefield A,
Ghazi A, Gerken C, Yi Z, Ashoori A, Wu MF, Liu H,
Rooney C, Dotti G, Gee A, Su J, Kew Y, Baskin D, Zhang
YJ, New P, Grilley B, Stojakovic M, Hicks J, Powell SZ,
Brenner MK, Heslop HE, Grossman R, Wels WS, andGottschalk S: HER2-specific chimeric antigen receptormodified virus-specific T cells for progressive glioblastoma:
A phase 1 dose-escalation trial. JAMA Oncol 2017;3:
1094–1101.

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

AN ANTI-HER2 MAB FOR GLIOBLASTOMAS

7. Zhang L, Ren J, Zhang H, Cheng G, Xu Y, Yang S, Dong
C, Fang D, Zhang J, and Yang A: HER2-targeted recombinant protein immuno-caspase-6 effectively induces apoptosis in HER2-overexpressing GBM cells in vitro andin vivo. Oncol Rep 2016;36:2689–2696.
8. Hegde M, Mukherjee M, Grada Z, Pignata A, Landi D,
Navai SA, Wakefield A, Fousek K, Bielamowicz K, Chow
KK, Brawley VS, Byrd TT, Krebs S, Gottschalk S, Wels
WS, Baker ML, Dotti G, Mamonkin M, Brenner MK,
Orange JS, and Ahmed N: Tandem CAR T cells targeting
HER2 and IL13Ralpha2 mitigate tumor antigen escape.
J Clin Invest 2016;126:3036–3052.
9. Takei J, Kaneko M, Ohishi T, Kawada M, Harada H,
and Kato Y: H2Mab-19, an anti-human epidermal growth
factor receptor 2 monoclonal antibody exerts antitumor
activity in mouse oral cancer xenografts. Exp Ther Med
2020;20:846–853.
10. Itai S, Fujii Y, Kaneko MK, Yamada S, Nakamura T,
Yanaka M, Saidoh N, Chang YW, Handa S, Takahashi M,
Suzuki H, Harada H, and Kato Y: H2Mab-77 is a sensitiveand specific anti-HER2 monoclonal antibody against breast
cancer. Monoclon Antib Immunodiagn Immunother 2017;36:143–148.
11. Kato Y, Kunita A, Abe S, Ogasawara S, Fujii Y, Oki H,
Fukayama M, Nishioka Y, and Kaneko MK: The chimericantibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather
than via its neutralizing activity. Oncotarget 2015;6:36003–
36018.
12. Howlader N, Altekruse SF, Li CI, Chen VW, Clarke CA,
Ries LA, and Cronin KA: US incidence of breast cancer
subtypes defined by joint hormone receptor and HER2
status. J Natl Cancer Inst 2014;106:dju055.
13. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A,
and McGuire WL: Human breast cancer: Correlation of
relapse and survival with amplification of the HER-2/neu
oncogene. Science 1987;235:177–182.
14. Xie S, Zhang H, Wang X, Ge Q, and Hu J: The relative
efficacy and safety of targeted agents used in combination
with chemotherapy in treating patients with untreatedadvanced gastric cancer: A network meta-analysis. Oncotarget 2017;8:26959–26968.
15. Lambert JM, and Chari RV: Ado-trastuzumab Emtansine(T-DM1): An antibody-drug conjugate (ADC) for HER2positive breast cancer. J Med Chem 2014;57:6949–6964.
16. Valabrega G, Montemurro F, and Aglietta M: Trastuzumab:
Mechanism of action, resistance and future perspectives in
HER2-overexpressing breast cancer. Ann Oncol 2007;18:
977–984.
17. Amiri-Kordestani L, Wedam S, Zhang L, Tang S, Tilley A,
Ibrahim A, Justice R, Pazdur R, and Cortazar P: First FDA
approval of neoadjuvant therapy for breast cancer: Pertuzumab for the treatment of patients with HER2-positive
breast cancer. Clin Cancer Res 2014;20:5359–5364.
18. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V,
Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram
M, Baselga J, and Norton L: Use of chemotherapy plus a
monoclonal antibody against HER2 for metastatic breast
cancer that overexpresses HER2. N Engl J Med 2001;344:
783–792.
19. Swain SM, Kim SB, Cortes J, Ro J, Semiglazov V,
Campone M, Ciruelos E, Ferrero JM, Schneeweiss A,

139

Knott A, Clark E, Ross G, Benyunes MC, and Baselga J:
Pertuzumab, trastuzumab, and docetaxel for HER2-positive
metastatic breast cancer (CLEOPATRA study): Overall
survival results from a randomised, double-blind, placebocontrolled, phase 3 study. Lancet Oncol 2013;14:461–471.
20. Doi T, Shitara K, Naito Y, Shimomura A, Fujiwara Y,
Yonemori K, Shimizu C, Shimoi T, Kuboki Y, Matsubara
N, Kitano A, Jikoh T, Lee C, Fujisaki Y, Ogitani Y, YverA, and Tamura K: Safety, pharmacokinetics, and antitumour activity of trastuzumab deruxtecan (DS-8201), a
HER2-targeting antibody-drug conjugate, in patients withadvanced breast and gastric or gastro-oesophageal tumours:
A phase 1 dose-escalation study. Lancet Oncol 2017;18:
1512–1522.
21. Nakada T, Sugihara K, Jikoh T, Abe Y, and Agatsuma T:
The latest research and development into the antibody-drug
conjugate, [fam-] trastuzumab deruxtecan (DS-8201a), for
HER2 cancer therapy. Chem Pharm Bull (Tokyo) 2019;67:
173–185.
22. Kaneko MK, Yamada S, Itai S, and Kato Y: Developmentof an anti-HER2 monoclonal antibody H2Mab-139 againstcolon cancer. Monoclon Antib Immunodiagn Immunother2018;37:59–62.
23. Yamada S, Itai S, Nakamura T, Chang YW, Harada H,
Suzuki H, Kaneko MK, and Kato Y: Establishment of
H2Mab-119, an anti-human epidermal growth factor receptor 2 monoclonal antibody, against pancreatic cancer.
Monoclon Antib Immunodiagn Immunother 2017;36:287–
290.
24. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
25. Ogasawara S, Kaneko MK, and Kato Y: LpMab-19
recognizes sialylated O-glycan on Thr76 of human podoplanin. Monoclon Antib Immunodiagn Immunother 2016;35:245–253.
26. Roberts M, Sevastou I, Imaizumi Y, Mistry K, Talma S,
Dey M, Gartlon J, Ochiai H, Zhou Z, Akasofu S, Tokuhara
N, Ogo M, Aoyama M, Aoyagi H, Strand K, Sajedi E,
Agarwala KL, Spidel J, Albone E, Horie K, Staddon JM,
and de Silva R: Pre-clinical characterisation of E2814, ahigh-affinity antibody targeting the microtubule-binding
repeat domain of tau for passive immunotherapy in Alzheimer’s disease. Acta Neuropathol Commun 2020;8:13.
27. Wang Y, Meng Y, Zhang S, Wu H, Yang D, Nie C, and Hu
Q: Phenformin and metformin inhibit growth and migrationof LN229 glioma cells in vitro and in vivo. Onco Targets
Ther 2018;11:6039–6048.

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 16, 2020
Accepted: June 14, 2020