MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 1, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0022
Open camera or QR reader andscan code to access this article
and other resources online.

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

Defucosylated Mouse-Dog Chimeric Anti-Human Epidermal
Growth Factor Receptor 2 Monoclonal Antibody (H77Bf)
Exerts Antitumor Activities in Mouse Xenograft Modelsof Canine Osteosarcoma
Ren Nanamiya,1,* Tomokazu Ohishi,2,* Hiroyuki Suzuki,3 Takuya Mizuno,4 Takeo Yoshikawa,5 Teizo Asano,1Tomohiro Tanaka,1 Mika K. Kaneko,1 and Yukinari Kato1,3,5

Human epidermal growth factor receptor 2 (HER2) has been studied in many human cancer types, and its
overexpression and/or gene mutation contribute to the poor prognosis. Therefore, HER2 is an importanttherapeutic target in various cancer types, including breast and gastric cancers. We previously developed ananti-HER2 monoclonal antibody (mAb), H2Mab-77 (mouse IgG1, kappa), which detects HER2 and dog HER2(dHER2) with high sensitivity and specificity. In this study, we produced a defucosylated mouse-dog chimericanti-HER2 mAb (H77Bf), and investigated the reactivity against canine osteosarcoma D-17 cells by flow
cytometry. Furthermore, we showed that H77Bf exerted antibody-dependent cellular cytotoxicity andcomplement-dependent cytotoxicity against D-17 cells in vitro and exhibited the potent antitumor activity
in vivo. These results suggest that H77Bf exerts antitumor effects against dHER2-expressing canine tumors andcould be valuable as part of an antibody treatment regimen for them.
Keywords: HER2, mouse-dog chimeric antibody, ADCC, CDC, canine osteosarcoma, antitumor activity

Trastuzumab, a monoclonal antibody (mAb) targeting
HER2, has improved survival rates in HER2-positive advanced and early-stage breast cancer, and has been the mosteffective therapy for HER2-positive breast cancer.11–15 The
recently developed trastuzumab deruxtecan (DS-8201) contains a novel enzyme cleavable-linker and a topoisomerase I
inhibitor, deruxtecan as a payload.16 Although there is noestablished regimen for patients with HER2-positive nonsmall cell lung cancer (NSCLC), the promising efficacy of
DS-8201 in patients with HER2-mutant NSCLC has beenreported.17
HER2 is also overexpressed in multiple canine cancers,
including OS,18 anal sac gland carcinoma,19 mammary carcinoma,20 and bladder transitional cell carcinoma.21 Fazekas

IntroductionHuman epidermal growth factor receptor 2(HER2) is a type-I transmembrane glycoprotein andmember of receptor tyrosine kinases. HER2 overexpression isreported in many human cancers, including breast cancer,1,2
gastric cancer,3 colorectal cancer,4 lung adenocarcinoma,5 andosteosarcoma (OS).6–8 Overexpression of HER2 results inhomo- or heterodimerization of the HER family members,
which leads to autophosphorylation of tyrosine residues in thecytoplasmic domains. The tyrosine phosphorylation initiates
signaling transductions including Ras-MEK-ERK and PI3
kinase-AKT pathways, which play critical roles in tumor cellproliferation, invasion, and tumorigenesis.9,10
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Shizuoka, Japan.
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
4
Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of Veterinary Medicine, Yamaguchi University,
Yamaguchi, Japan.
5
Department of Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
*Contributed equally to this study.
2
3

27

28

et al conducted a clinical trial using the HER2 vaccine in aspontaneous HER2-positive canine mammary carcinoma toevaluate the safety and efficacy.22 In a phase I study in canineOS, administration of a Listeria vaccine expressing chimeric
HER2 induced dog HER2 (dHER2)-specific immunity,
suppressed the development of metastases, and prolonged
survival.23 Therefore, dHER2 could be a promising therapeutic treatment of canine cancers.
We previously developed anti-HER2 mAbs, including
H2Mab-19,24–27 H2Mab-41,28,29 H2Mab-77,30 H2Mab-119,31
H2Mab-139,32 and H2Mab-181,33,34 which detect not only
HER2 but also dHER2. This study aimed to investigate theability of a defucosylated mouse-dog chimeric H2Mab-77(H77Bf) to induce antibody-dependent cellular cytotoxicity(ADCC), complement-dependent cytotoxicity (CDC), andantitumor activities against a canine OS cell line, which endogenously expresses dHER2.

NANAMIYA ET AL.

maximum tumor size >2000 mm3 as humane endpoints foreuthanasia. Mice were euthanized by cervical dislocation;
death was confirmed by respiratory and cardiac arrest.
Flow cytometry

D-17 cells were harvested by brief exposure to 0.25%
trypsin/1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). After washing with blocking buffer of
0.1% bovine serum albumin in phosphate-buffered saline(PBS), cells were treated with H77Bf or control blocking
buffer for 30 minutes at 4C. Subsequently, the cells wereincubated in fluorescein isothiocyanate (FITC)-conjugatedanti-dog IgG at a dilution of 1:1000 (Thermo Fisher ScientificInc.) for 30 minutes at 4C. Fluorescence data were collectedusing the EC800 Cell Analyzer (Sony Corp., Tokyo, Japan).

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

Determination of the binding affinity
Materials and Methods
Cell lines

D-17, a canine OS cell line,35,36 was obtained from theAmerican Type Culture Collection (Manassas, VA). D-17
was cultured in minimum essential media (Nacalai Tesque,Inc., Kyoto, Japan), supplemented with 10% heatinactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA), 1 mM of sodium pyruvate,
100 U/mL of penicillin, 100 lg/mL streptomycin, and0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.). The
cells were cultured at 37C in a humidified atmosphere with
5% carbon dioxide.
Antibodies

An anti-HER2 mAb, H2Mab-77, was established in ourlaboratory as previously described.30 To generate H77B, we
subcloned VH cDNA of H2Mab-77 and CH of dog IgGB into
the pCAG-Ble vector (FUJIFILM Wako Pure Chemical
Corporation, Osaka, Japan), along with VL cDNA of H2Mab77 and CL cDNA of dog kappa light chain into the pCAGNeo vector (FUJIFILM Wako Pure Chemical Corporation),
respectively. The vector of H77B was transfected into
BINDS-09 cells (FUT8-deficient ExpiCHO-S cells)37–43 using the ExpiCHO Expression System (Thermo Fisher Scientific Inc.). The resulting mAb, H77Bf, was purified withProtein G-Sepharose (GE Healthcare Biosciences, Pittsburgh, PA). Dog IgG was commercially purchased from
Jackson ImmunoResearch Inc. (West Grove, PA).
Animals

All animal experiments were carried out following relevant guidelines and regulations to minimize animal sufferingand distress in the laboratory. Animal experiments for testing
antitumor activity were approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (permit no. 2021-056). Mice were kept in a specificpathogen-free environment (23C – 2C, 55% – 5% humidity) on an 11-hour light/13-hour dark cycle with food andwater supplied ad libitum across the experimental period.
Mice were monitored for health and weight every 2–5 days
during the 3-week period of each experiment. We determined
the loss of original body weight to a point >25% and/or a

D-17 cells were suspended in 100 lL of serially diluted
H77Bf (0.006–25 lg/mL) followed by FITC-conjugated antidog IgG (1:200). Fluorescence data were collected using theEC800 Cell Analyzer. The dissociation constant (KD) was
calculated by fitting binding isotherms to built-in one-sitebinding models in GraphPad Prism 8 (GraphPad Software,Inc., La Jolla, CA).
ADCC of H77Bf

Canine mononuclear cells (MNCs) were obtained from
Yamaguchi University and suspended in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque, Inc.) with 10%
FBS to be used as effector cells. Target cells were labeledwith 10 lg/mL Calcein AM (Thermo Fisher Scientific, Inc.)and resuspended in the same medium. The target cells(2 · 104 cells/well) were plated in 96-well plates and mixed
with effector cells (effector/target cells ratio, 50), 100 lg/mLof H77Bf or control dog IgG. After 4.5 hours incubation at
37C, the release of Calcein into the supernatant was measured in each well.
The fluorescence intensity was determined using a microplate reader (Power Scan HT; BioTek Instruments, Inc.,
Winooski, VT) with an excitation wavelength of 485 nm andan emission wavelength of 538 nm. Cytolytic activity (%lysis) was calculated as follows: % lysis = (E - S)/(M - S) ·100, where ‘‘E’’ is the fluorescence measured in combined
cultures of target and effector cells, ‘‘S’’ is the spontaneous
fluorescence of target cells only, and ‘‘M’’ is the maximum
fluorescence measured after the lysis of all cells with a buffer
containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4),
and 10 mM EDTA.
CDC of H77Bf

The target cells were labeled with 10 lg/mL Calcein AM
and resuspended in the medium. Then, they were plated in
96-well plates at 2 · 104 cells/well with rabbit complement(final dilution 1:10; Low-Tox-M Rabbit Complement; Cedarlane Laboratories, Hornby, Ontario, Canada) and
100 lg/mL of H77Bf or control dog IgG. After 4.5 hours of
incubation at 37C, we measured Calcein release into thesupernatant for each well. Fluorescence intensity was calculated as described in the ADCC section earlier.

DEFUCOSYLATED ANTI-HER2 MAB

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

Antitumor activity of H77Bf in mouse xenograft modelsof D-17 cells

A total of 16 female BALB/c nude mice (5 weeks old,weighing 14–17 g) were purchased from Charles River Laboratories, Inc. and used in experiments once they reached 8
weeks of age. D-17 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);
100 lL of this suspension (5 · 106 cells) was injected subcutaneously into the left flanks of the mice. On day 8 postinoculation, 100 lg of H77Bf (n = 8) or control dog IgG(n = 8) in 100 mL PBS were injected intraperitoneally. Additional antibody inoculations were performed on days 14 and21. MNCs were injected surrounding the tumors on days 8,
14, and 21. The tumor volume was measured on days 7, 10,
14, 17, 21, 24, and 28 after the injection of cells. At 28 days
after cell implantation, all mice were euthanized by cervicaldislocation. Tumor diameters and volumes were measured aspreviously described.35,36,44,45

29
Statistical analyses

All data are expressed as mean – standard error of
the mean. All statistical analyses were carried out with
Welch’s t-test for ADCC, CDC, and tumor weight. Sidak’s
multiple comparisons tests were conducted for tumor volume
and mouse weight. All calculations were performed using
GraphPad Prism 8. A p-value <0.05 is statistically significant.
Results
Flow cytometric analysis against canine OS D-17 cells
using H77Bf

An anti-HER2 mAb, H2Mab-77, was previously establishedby immunizing mice with ectodomain of HER2.30 We next
generated H77Bf, a defucosylated mouse-dog chimeric antiHER2 mAb by combining VH and VL of H2Mab-77 with CH
and CL of dog IgG, respectively. We investigated the reactivityof H77Bf against canine OS D-17 cells, and found that H77Bf
could detect endogenous dHER2 in D-17 cells (Fig. 1A).

FIG. 1. Flow cytometry using H77Bf and evaluation of ADCC and CDC. (A) D-17 cells were treated with H77Bf (10–0.01 mg/mL), followed by secondary antibodies. (B) Determination of the binding affinity of H77Bf for D-17 cells using
flow cytometry. D-17 cells were suspended in 100 mL of serially diluted H77Bf, followed by the addition of FITCconjugated anti-dog IgG. Fluorescence data were collected using the EC800 Cell Analyzer. (C, D) ADCC (C) and CDC(D) elicited by H77Bf and control dog IgG targeting D-17 cells. The values shown are the means – SEM. Asterisks indicate
statistical significance (*p < 0.05; Welch’s t-test). ADCC, antibody-dependent cellular cytotoxicity; CDC, complementdependent cytotoxicity; FITC, fluorescein isothiocyanate; SEM, standard error of the mean.

30

A kinetic analysis of the interactions of H77Bf with D-17 cells
was carried out using flow cytometry. The KD for the interactionof H77Bf with D-17 cells was 6.7 · 10-10 M (Fig. 1B), demonstrating that H77Bf has high affinity for D-17 cells.
H77Bf-mediated ADCC and CDC in D-17 cells

We investigated whether H77Bf mediated ADCC against
D-17 cells. As shown in Figure 1C, H77Bf showed ADCC
(10.4% cytotoxicity; p < 0.05) against D-17 cells more effectively than did the control dog IgG (3.4% cytotoxicity).

NANAMIYA ET AL.

Subsequently, we investigated whether H77Bf mediated
CDC against D-17 cells. As shown in Figure 1D, H77Bf
showed CDC (68.4% cytotoxicity; p < 0.05) against D-17
cells more highly than did the control dog IgG (53.3% cytotoxicity). These results indicated that H77Bf promoted
significantly higher levels of ADCC and CDC against endogenous dHER2-expressing D-17 cells.
Antitumor activities of H77Bf in the mouse xenograftmodels of D-17 cells

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

In the D-17 mouse xenograft models, H77Bf (100 lg) andcontrol dog IgG (100 lg) were injected intraperitoneally intomice on days 8, 14, and 21, after the injection of D-17 cells.
The tumor volume was measured on days 7, 10, 14, 17, 21,
24, and 28 after the injection of the cells. H77Bf treatment
significantly suppressed tumor development on days 14( p < 0.05), 17 ( p < 0.01), 21 ( p < 0.01), 24 ( p < 0.01), and 28( p < 0.01) compared with that of the control dog IgG(Fig. 2A). The administration of H77Bf resulted in a 62%
reduction of tumor mass compared with that of the control
dog IgG on day 28 postinoculation. Furthermore, tumors

FIG. 2. Evaluation of antitumor activity of H77Bf in D-17
xenograft models. (A) D-17 cells (5 · 106 cells) were injected subcutaneously into the left flank. On day 8, 100 mg of
H77Bf or control dog IgG in 100 mL PBS were injected
intraperitoneally into mice; additional antibodies were theninjected on days 14 and 21. The tumor volume was measured on days 7, 10, 14, 17, 21, 24, and 28 after the injection. Values shown are the means – SEM. Asterisks indicate
statistical significance (**p < 0.01, *p < 0.05; ANOVA andSidak’s multiple comparisons test). (B) Tumors of D-17
xenografts were resected from H77Bf and control dog IgGgroups. Tumor weight on day 28 was measured from excised xenografts. Values are mean – SEM. Asterisk indicatesstatistical significance (**p < 0.01, Welch’s t-test).
(C) Resected tumors of D-17 xenografts from control dog
IgG and H77Bf groups on day 28. Scale bar, 1 cm. ANOVA,
analysis of variance; PBS, phosphate-buffered saline.

FIG. 3. Body weights and appearance of the mice implanted with D-17 xenograft models. (A) Body weights ofthe mice implanted with D-17 xenograft models were recorded on days 7, 10, 14, 17, 21, 24, and 28. (B) Body
appearance of mice on day 28. Values shown are the means– SEM. Scale bar, 1 cm. n.s., not significant.

DEFUCOSYLATED ANTI-HER2 MAB

from the H77Bf-treated mice weighed significantly less thanthose from the control dog IgG-treated mice (60% reduction;p < 0.01, Fig. 2B). Tumors removed from mice on day 28 areshown in Figure 2C. Total body weights did not differ significantly among the two groups (Fig. 3A). The body appearance of mice on day 28 are shown in Figure 3B.
Taken together, these results indicated that the administration of H77Bf effectively reduced the growth of D-17
xenografts.

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

Discussion

OS is the most frequent primary bone cancer in dogs.
Eighty percent of dogs with OS is lethal due to the lungmetastasis.46 Selvarajah et al reported that the epidermal
growth factor receptor (EGFR) expression was significantly
upregulated in OS lung metastasis compared with extrapulmonary sites.47 The rate of OS in dogs is 27 times higher
than that in humans, with a 1-year survival rate of only
45%.48 Currently, treatment options for OS in dogs include
surgery (limb amputation or limb-sparing surgery), radiation therapy, and chemotherapy.49,50 However, satisfied
therapeutic efficacies have not been obtained. Hence, thedevelopment of other therapeutic options for canine OS isnecessary.
Antibody therapy is widely used for human diseases including cancers and immune disorders. The caninization ofmAbs such as Lokivetmab51 is essential for developing antibody therapies for dogs. However, caninized mAbs for canine
cancers have not been developed yet. Thus, the developmentof the mAbs for canine cancer treatment is needed.
The amino acid sequences are similar between human anddog about EGFR and HER2; in contrast, low homology wasobserved between human and dog about CD20 andprogrammed-cell death ligand 1.52–54 Therefore, anti-human
EGFR or anti-HER2 mAbs could be applicable to canine
cancers. Indeed, cetuximab (anti-human EGFR mAb) andtrastuzumab exhibited growth inhibitory effect by interferingdog EGFR (dEGFR) and dHER2 signaling, respectively.52
We also have been investigating the cross-reactivity of ourestablished anti-human EGFR mAb (EMab-134) todEGFR35,36,44,45 and another anti-HER2 mAb (H2Mab-41) todHER2.29
Our study demonstrated that H77Bf significantly exhibited
ADCC and CDC activities in canine OS D-17 cells comparedwith dog IgG in vitro (Fig. 1C, D). One of the most important
objectives of this study was to investigate the antitumor
activity of H77Bf for endogenous dHER2-expressing canine tumor cells in vivo. H77Bf administration showed thepotent antitumor effect on endogenous dHER2-expressing
D-17 cells without body weight loss and skin disorders(Figs. 2 and 3).
These results indicate that H77Bf could be useful for an
antibody therapeutic regimen for dHER2-positive canine OS.
Moreover, H2Mab-77, which is the origin of H77Bf, is applicable for immunohistochemistry30; therefore, we need toinvestigate whether H77Bf detects endogenous dHER2 incanine OS tissues to evaluate usefulness of H77Bf as a diagnostic mAb for immunohistochemistry. Future studies arenecessary to test the antitumor activity of H77Bf on otherseveral types of endogenous dHER2-expressing canine cancers in vivo animal experimental models.

31
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: JP22ama121008 (to Y.K.), JP22am0401013 (toY.K.), JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.),
and JP21am0101078 (to Y.K.), and by the Japan Society for
the Promotion of Science (JSPS) Grants-in-Aid for Scientific
Research (KAKENHI) grant nos. 22K15523 (to T.A.), 21K
20789 (to T.T.), 22K06995 (to H.S.), 22K07168 (to M.K.K.),
and 22K07224 (to Y.K.).
References

1. Howlader N, Altekruse SF, Li CI, et al. US incidence ofbreast cancer subtypes defined by joint hormone receptorand HER2 status. J Natl Cancer Inst 2014;106(5):dju055;
doi: 10.1093/jnci/dju055
2. Slamon DJ, Clark GM, Wong SG, et al. Human breast
cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177–
182; doi: 10.1126/science.3798106
3. Xie S, Zhang H, Wang X, et al. The relative efficacy andsafety of targeted agents used in combination with chemotherapy in treating patients with untreated advanced
gastric cancer: A network meta-analysis. Oncotarget 2017;
8:26959–26968; doi: 10.18632/oncotarget.15923
4. Seo AN, Kwak Y, Kim DW, et al. HER2 status in colorectal cancer: Its clinical significance and the relationshipbetween HER2 gene amplification and expression. PLoS
One 2014;9:e98528; doi: 10.1371/journal.pone.0098528
5. Kim EK, Kim KA, Lee CY, et al. The frequency andclinical impact of HER2 alterations in lung adenocarcinoma. PLoS One 2017;12:e0171280; doi: 10.1371/journal.pone.0171280
6. Onda M, Matsuda S, Higaki S, et al. ErbB-2 expression iscorrelated with poor prognosis for patients with osteosarcoma. Cancer 1996;77:71–78; doi: 10.1002/(SICI)10970142(19960101)77:1 <71::AID-CNCR13>3.0.CO;2–5
7. Gorlick R, Huvos AG, Heller G, et al. Expression of
HER2/erbB-2 correlates with survival in osteosarcoma.
J Clin Oncol 1999;17:2781–2788; doi: 10.1200/JCO.1999.17.9.2781
8. Scotlandi K, Manara MC, Hattinger CM, et al. Prognostic
and therapeutic relevance of HER2 expression in osteosarcoma and Ewing’s sarcoma. Eur J Cancer 2005;41:
1349–1361; doi: 10.1016/j.ejca.2005.03.015
9. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling
network. Nat Rev Mol Cell Biol 2001;2:127–137; doi:
10.1038/35052073
10. Krishnamurti U, Silverman JF. HER2 in breast cancer: A
review and update. Adv Anat Pathol 2014;21:100–107; doi:
10.1097/PAP.0000000000000015
11. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med
2001;344:783–792; doi: 10.1056/NEJM200103153441101
12. Slamon D, Eiermann W, Robert N, et al. Adjuvant trastuzumab in HER2-positive breast cancer. N Engl J Med 2011;
365:1273–1283; doi: 10.1056/NEJMoa0910383
13. Smith I, Procter M, Gelber RD, et al. 2-year follow-up of
trastuzumab after adjuvant chemotherapy in HER2-positiveDownloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

32

breast cancer: A randomised controlled trial. Lancet 2007;
369:29–36; doi: 10.1016/S0140-6736(07)60028-2
14. Okines AF, Cunningham D. Trastuzumab: A novel standard
option for patients with HER-2-positive advanced gastric or
gastro-oesophageal junction cancer. Therap Adv Gastroenterol 2012;5:301–318; doi: 10.1177/1756283X12450246
15. Oh DY, Bang YJ. HER2-targeted therapies - a role beyond
breast cancer. Nat Rev Clin Oncol 2020;17:33–48; doi:
10.1038/s41571-019-0268-3
16. Doi T, Shitara K, Naito Y, et al. Safety, pharmacokinetics,
and antitumour activity of trastuzumab deruxtecan (DS8201), a HER2-targeting antibody-drug conjugate, in patients with advanced breast and gastric or gastro-oesophageal
tumours: A phase 1 dose-escalation study. Lancet Oncol
2017;18:1512–1522; doi: 10.1016/S1470-2045(17)30604-6
17. Li BT, Smit EF, Goto Y, et al. Trastuzumab deruxtecan in
HER2-mutant non-small-cell lung cancer. N Engl J Med
2022;386:241–251; doi: 10.1056/NEJMoa2112431
18. Flint AF, U’Ren L, Legare ME, et al. Overexpression of theerbB-2 proto-oncogene in canine osteosarcoma cell lines
and tumors. Vet Pathol 2004;41:291–296; doi:
10.1354/vp.41-3-291
19. Yoshimoto S, Kato D, Kamoto S, et al. Detection of humanepidermal growth factor receptor 2 overexpression in canine anal sac gland carcinoma. J Vet Med Sci 2019;81:1034–1039; doi: 10.1292/jvms.19-0019
20. Gama A, Alves A, Schmitt F. Identification of molecular phenotypes in canine mammary carcinomas with clinical implications: Application of the human classification. VirchowsArch 2008;453:123–132; doi: 10.1007/s00428-008-0644-3
21. Millanta F, Impellizeri J, McSherry L, et al. Overexpression of HER-2 via immunohistochemistry in canine
urinary bladder transitional cell carcinoma—A marker ofmalignancy and possible therapeutic target. Vet Comp
Oncol 2018;16:297–300; doi: 10.1111/vco.12345
22. Fazekas J, Furdos I, Singer J, et al. Why man’s best friend,
the dog, could also benefit from an anti-HER-2 vaccine.
Oncol Lett 2016;12:2271–2276; doi: 10.3892/ol.2016.5001
23. Mason NJ, Gnanandarajah JS, Engiles JB, et al. Immunotherapy with a HER2-targeting listeria induces HER2specific immunity and demonstrates potential therapeutic
effects in a phase I trial in canine osteosarcoma. Clin
Cancer Res 2016;22:4380–4390; doi: 10.1158/1078-0432.CCR-16-0088
24. Kato Y, Ohishi T, Sano M, et al. H2Mab-19 anti-humanepidermal growth factor receptor 2 monoclonal antibody
therapy exerts antitumor activity in pancreatic cancer xenograft models. Monoclon Antib Immunodiagn Immunother 2020;39:61–65; doi: 10.1089/mab.2020.0011
25. Kato Y, Ohishi T, Takei J, et al. An antihuman epidermal
growth factor receptor 2 monoclonal antibody (H2Mab-19)
exerts antitumor activity in glioblastoma xenograft models.
Monoclon Antib Immunodiagn Immunother 2020;39:135–
139; doi: 10.1089/mab.2020.0013
26. Kato Y, Ohishi T, Takei J, et al. An anti-human epidermal
growth factor receptor 2 monoclonal antibody H2Mab-19
exerts antitumor activity in mouse colon cancer xenografts.
Monoclon Antib Immunodiagn Immunother 2020;39:123–
128; doi: 10.1089/mab.2020.0009
27. Takei J, Kaneko MK, Ohishi T, et al. H2Mab-19, an antihuman epidermal growth factor receptor 2 monoclonal
antibody exerts antitumor activity in mouse oral cancer
xenografts. Exp Ther Med 2020;20:846–853; doi: 10.3892/
etm.2020.8765

NANAMIYA ET AL.

28. Kato Y, Ohishi T, Yamada S, et al. Anti-human epidermal
growth factor receptor 2 monoclonal antibody H2Mab-41
exerts antitumor activity in a mouse xenograft model ofcolon cancer. Monoclon Antib Immunodiagn Immunother2019;38:157–161; doi: 10.1089/mab.2019.0017
29. Tateyama N, Asano T, Ohishi T, et al. An anti-HER2
monoclonal antibody H2Mab-41 exerts antitumor activities
in mouse xenograft model using dog HER2-overexpressed
cells. Monoclon Antib Immunodiagn Immunother 2021;40:
184–190; doi: 10.1089/mab.2021.0025
30. Itai S, Fujii Y, Kaneko MK, et al. H2Mab-77 is a sensitiveand specific anti-HER2 monoclonal antibody against breast
cancer. Monoclon Antib Immunodiagn Immunother 2017;36:143–148; doi: 10.1089/mab.2017.0026
31. Yamada S, Itai S, Nakamura T, et al. 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; doi: 10.1089/mab.2017.0050
32. Kaneko MK, Yamada S, Itai S, et al. Development of ananti-HER2 monoclonal antibody H2Mab-139 against coloncancer. Monoclon Antib Immunodiagn Immunother 2018;37:59–62; doi: 10.1089/mab.2017.0052
33. Asano T, Takei J, Suzuki H, et al. Epitope mapping of ananti-HER2 monoclonal antibody (H2Mab-181) using
enzyme-linked immunosorbent assay. Monoclon AntibImmunodiagn Immunother 2021;40:255–260; doi: 10.1089/
mab.2021.0029
34. Takei J, Asano T, Tanaka T, et al. Development of a novelanti-HER2 monoclonal antibody H2Mab-181 for gastriccancer. Monoclon Antib Immunodiagn Immunother 2021;
40:168–176; doi: 10.1089/mab.2021.0021
35. Li G, Ohishi T, Kaneko MK, et al. Defucosylated mousedog chimeric anti-EGFR antibody exerts antitumor activities in mouse xenograft models of canine tumors. Cells
2021;10(12):3599; doi: 10.3390/cells10123599
36. Nanamiya R, Takei J, Ohishi T, et al. Defucosylated antiepidermal growth factor receptor monoclonal antibody (134mG2a-f) exerts antitumor activities in mouse xenograft models
of canine osteosarcoma. Monoclon Antib Immunodiagn Immunother 2022;41:1–7; doi: 10.1089/mab.2021.0036
37. Mizuno T, Kato Y, Kaneko MK, et al. Generation of acanine anti-canine CD20 antibody for canine lymphoma
treatment. Sci Rep 2020;10:11476; doi: 10.1038/s41598020-68470-9
38. Nanamiya R, Suzuki H, Takei J, et al. Development of
monoclonal antibody 281-mG2a-f against golden hamster
podoplanin. Monoclon Antib Immunodiagn Immunother2022; doi: 10.1089/mab.2021.0058
39. Kawabata H, Ohishi T, Suzuki H, et al. A defucosylatedmouse anti-CD10 monoclonal antibody (31-mG2a-f) exerts
antitumor activity in a mouse xenograft model of renal cell
cancers. Monoclon Antib Immunodiagn Immunother 2022;
doi: 10.1089/mab.2021.0049
40. Takei J, Kaneko MK, Ohishi T, et al. A defucosylated
antiCD44 monoclonal antibody 5mG2af exerts antitumor
effects in mouse xenograft models of oral squamous cell
carcinoma. Oncol Rep 2020;44:1949–1960; doi: 10.3892/or.2020.7735
41. Kawabata H, Suzuki H, Ohishi T, et al. A defucosylatedmouse anti-CD10 monoclonal antibody (31-mG2a-f) exerts
antitumor activity in a mouse xenograft model of CD10overexpressed tumors. Monoclon Antib Immunodiagn Immunother 2022;41:59–66; doi: 10.1089/mab.2021.0048

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

DEFUCOSYLATED ANTI-HER2 MAB

42. Li G, Suzuki H, Takei J, et al. Antitumor activities inmouse xenograft models of canine mammary gland tumor
by defucosylated mouse-dog chimeric anti-epidermal
growth factor receptor antibody (E134Bf). Monoclon AntibImmunodiagn Immunother 2022;41:53–58; doi: 10.1089/
mab.2021.0040
43. Takei J, Ohishi T, Kaneko MK, et al. A defucosylated antiPD-L1 monoclonal antibody 13-mG2a-f exerts antitumor
effects in mouse xenograft models of oral squamous cell
carcinoma. Biochem Biophys Rep 2020;24:100801; doi:
10.1016/j.bbrep.2020.100801
44. Goto N, Suzuki H, Ohishi T, et al. Antitumor activities inmouse xenograft models of canine fibroblastic tumor bydefucosylated anti-epidermal growth factor receptor
monoclonal antibody. Monoclon Antib Immunodiagn Immunother 2022;41:67–73; doi: 10.1089/mab.2021.0059
45. Tateyama N, Nanamiya R, Ohishi T, et al. Defucosylated
anti-epidermal growth factor receptor monoclonal antibody
134-mG2a-f exerts antitumor activities in mouse xenograftmodels of dog epidermal growth factor receptoroverexpressed cells. Monoclon Antib Immunodiagn Immunother 2021;40:177–183; doi: 10.1089/mab.2021.0022
46. Szewczyk M, Lechowski R, Zabielska K. What do we know
about canine osteosarcoma treatment? Review. Vet Res
Commun 2015;39:61–67; doi: 10.1007/s11259-014-9623-0
47. Selvarajah GT, Verheije MH, Kik M, et al. Expression of
epidermal growth factor receptor in canine osteosarcoma:
Association with clinicopathological parameters and prognosis. Vet J 2012;193:412–419; doi: 10.1016/j.tvjl.2012.02.009
48. Simpson S, Dunning MD, de Brot S, et al. Comparative
review of human and canine osteosarcoma: Morphology,
epidemiology, prognosis, treatment and genetics. Acta Vet
Scand 2017;59:71; doi: 10.1186/s13028-017-0341-9
49. Boston SE, Ehrhart NP, Dernell WS, et al. Evaluation ofsurvival time in dogs with stage III osteosarcoma that undergo
treatment: 90 cases (1985–2004). J Am Vet Med Assoc 2006;
228:1905–1908; doi: 10.2460/javma.228.12.1905

33

50. Selmic LE, Burton JH, Thamm DH, et al. Comparison ofcarboplatin and doxorubicin-based chemotherapy protocols
in 470 dogs after amputation for treatment of appendicularosteosarcoma. J Vet Intern Med 2014;28:554–563; doi:
10.1111/jvim.12313
51. Michels GM, Ramsey DS, Walsh KF, et al. A blinded,
randomized, placebo-controlled, dose determination trial of
lokivetmab (ZTS-00103289), a caninized, anti-canine IL31 monoclonal antibody in client owned dogs with atopic
dermatitis. Vet Dermatol 2016;27:478-e129; doi: 10.1111/
vde.12376
52. Singer J, Weichselbaumer M, Stockner T, et al. Comparative oncology: ErbB-1 and ErbB-2 homologues in canine
cancer are susceptible to cetuximab and trastuzumab targeting. Mol Immunol 2012;50:200–209; doi: 10.1016/j.molimm.2012.01.002
53. Dias JNR, Almeida A, Andre AS, et al. Characterization of
the canine CD20 as a therapeutic target for comparative
passive immunotherapy. Sci Rep 2022;12:2678; doi:
10.1038/s41598-022-06549-1
54. Maekawa N, Konnai S, Ikebuchi R, et al. Expression of PDL1 on canine tumor cells and enhancement of IFN-gamma
production from tumor-infiltrating cells by PD-L1 blockade. PLoS One 2014;9:e98415; doi: 10.1371/journal.pone.0098415

Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: May 24, 2022
Accepted: September 30, 2022