Biochemistry and Biophysics Reports 17 (2019) 23–26

Contents lists available at ScienceDirect

Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

The mouse–canine chimeric anti-dog podoplanin antibody P38B exerts
antitumor activity in mouse xenograft models

T⁎

Yukinari Katoa,b, , Tomokazu Ohishic, Manabu Kawadac, Naoya Maekawad, Satoru Konnaid,e,Shunsuke Itaia, Shinji Yamadaa, Mika K. Kanekoa
aDepartment of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapanNew Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japanc
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, 18-24 Miyamoto, Numazu-shi, Shizuoka 410-0301, JapandDepartment of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo 060-0818, Japane
Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo 060-0818,
JapanbA R T I C LE I N FO

A B S T R A C T

Keywords:
Mouse-canine chimeric antibody
Dog podoplanin
dPDPN
Monoclonal antibody

Podoplanin (PDPN) is a type I transmembrane heavily glycosylated sialoglycoprotein that is expressed in normal
tissues such as pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells. PDPN overexpression in cancerous tissue is associated with hematogenous metastasis through interactions with the C-type
lectin-like receptor 2 (CLEC-2). Previously, we have reported the development of a mouse monoclonal antibody(mAb), PMab-38 (IgG1, kappa) against dog PDPN (dPDPN). PMab-38 was found to strongly react with canine
squamous cell carcinomas (SCCs) and melanomas; however, it showed no reaction with lymphatic endothelial
cells. Recently, we have developed and produced the mouse–canine mAb of subclass B, P38B that showed antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against Chinese hamster
ovary (CHO)/dPDPN cells. In the present study, we investigated the antitumor activity using mouse xenograftmodel. To induce ADCC activity by P38B, canine mononuclear cells were injected surrounding the tumors in axenograft model. It was demonstrated that P38B exerted antitumor activity against the mouse xenograft modelusing CHO/dPDPN. These results suggest that P38B is useful for antibody therapy against dPDPN-expressing
canine SCCs and melanomas.

1. Introduction
Podoplanin (PDPN) is expressed in normal tissue, including pulmonary type I alveolar cells, renal podocytes, chondrocytes, myoﬁbroblasts, mesothelial cells, and lymphatic endothelial cells [1]. PDPN
overexpression is also observed in diﬀerent types of tumors, including
mesotheliomas [2,3], squamous cell carcinomas (SCC) [4], testiculartumors [5], and glioblastomas [6]. Recent clinical studies have provided evidence for the association between increased PDPN expression
and poor disease prognosis [7]. This observation indicated that theestablishment of anti-PDPN monoclonal antibodies (mAbs) is critical fordeveloping novel therapeutic strategies against cancer development
and metastatic progression [8].
Dog PDPN (dPDPN) was previously reported in the literature as

gp40 [9]. We have recently developed two mAbs PMab-38 (mouse IgG1,kappa) [10] and PMab-48 (mouse IgG1, kappa) [11]. It was shown that
PMab-38 recognized dPDPN of renal epithelial cells weakly but showedno reaction with lymphatic endothelial cells [10]. Conversely, PMab-48
reacted with renal epithelial cells as well as with lymphatic endothelial
cells [11]. Tyr67 and Glu68 of dPDPN were determined as the critical
epitopes of PMab-38 [12], whereas Asp29, Asp30, Ile31, Ile32, andPro33 of dPDPN were essential for the recognition of PMab-48 [13].
Immunohistochemistry demonstrated that PMab-38 reacted with 83%of canine SCCs (15/18 cases) [14] and 90% of melanomas (9/10 cases)[15]. Recently, we have developed a mouse–canine chimeric antibody(P38B) that was derived from PMab-38 [16]. P38B demonstrated antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against CHO/dPDPN cells, indicating thatAbbreviations: ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; CHO, Chinese hamster ovary; CLEC-2, C-type lectin-likereceptor 2; dPDPN, dog podoplanin; mAb, monoclonal antibody; PBS, phosphate-buﬀered saline; PDPN, podoplanin; SCC, squamous cell carcinomas⁎
Corresponding author at: Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai,
Miyagi 980-8575, Japan.
E-mail addresses: yukinarikato@med.tohoku.ac.jp, yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2018.11.005
Received 24 October 2018; Accepted 14 November 20182405-5808/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license(http://creativecommons.org/licenses/BY/4.0/).

Biochemistry and Biophysics Reports 17 (2019) 23–26

Y. Kato et al.

targeting cancer cells without adverse events.
To date, few studies have been performed on the subclasses (A, B, C,D) of canine IgGs [22,23]. However, it was demonstrated that the canine subclasses A and D appear to be eﬀector-function negative,
whereas subclasses B and C bind to canine Fc gamma receptors and are
positive for ADCC similar to human IgG1 and IgG3, respectively [22].
Furthermore, subclasses B and C can induce CDC. An anti-canine CD20mAb (1E4) was established to treat canine B-cell lymphoma and produced mouse–canine chimeric antibodies [23]. It was also shown that
1E4-cIgGB (subclass B) and 1E4-cIgGC (subclass C) led to signiﬁcantdepletion of B-cell levels in healthy beagle dogs. Moreover, we produced P38B, a mouse–canine chimeric antibody of canine subclass B,
from PMab-38 [16]. In that study, we clearly demonstrated that P38B
possesses ADCC and CDC against CHO/dPDPN cells.
To study the antitumor activity of P38B on cell growth in vivo,
CHO/dPDPN cells or CHO-K1 cells were subcutaneously implanted intothe ﬂanks of nude mice. P38B and control dog IgG were injected threetimes (on day 1, 8, and 15 after cell injections) into the peritoneal
cavity of mice. Canine mononuclear cells were injected three times (onday 1, 8, and 15) surrounding the xenograft. Tumor formation wasobserved in mice from the control and P38B-treated groups in CHO/dPDPN xenograft models (Fig. 1A) and CHO-K1 xenograft models(Fig. 1B). However, P38B signiﬁcantly reduced tumor development ofCHO/dPDPN xenograft compared with control dog IgG on day 14 andP38B is applicable for antibody-based therapy for canine cancers. In thepresent study, we investigated antitumor activity against mouse xenograft models using CHO/dPDPN and CHO-K1.
2. Materials and methods
2.1. Cell lines
Chinese hamster ovary (CHO)-K1 cell-line was obtained from theAmerican Type Culture Collection (ATCC; Manassas, VA). In our previous studies, we have inserted dPDPN with a N-terminal PA tag and a
C-terminal RAP tag-MAP tag (PA-dPDPN-RAP-MAP) in a pCAG-Blevector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)[10]. The PA tag [17], RAP tag [18], and MAP tag [19] comprises 12
amino acids each—GVAMPGAEDDVV, DMVNPGLEDRIE, and GDGMVPPGIEDK, respectively. CHO-K1 cells were transfected with pCAGBle/PA-dPDPN-RAP-MAP using Gene Pulser Xcell electroporationsystem (Bio-Rad Laboratories Inc., Berkeley, CA) resulting in the cellline CHO/dPDPN. CHO-K1 and CHO/dPDPN were cultured in RPMI
1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with
10% heat-inactivated fetal bovine serum (Thermo Fisher Scientiﬁc Inc.,Waltham, MA), 100 units/mL of penicillin, 100 μg/mL of streptomycin,
and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37 °C in ahumidiﬁed atmosphere of 5% CO2 and 95% air.
2.2. Antibodies
The mouse anti-dPDPN mAb PMab-38 was developed as previouslydescribed [10]. To generate the mouse–canine (subclass B) chimericantibody P38B, the appropriate VH and VL cDNAs of mouse PMab-38
and the CH and CL of canine IgG subclass B were subcloned into pCAGBle and pCAG-Neo vectors (FUJIFILM Wako Pure Chemical Corporation), respectively [16]. Expression vectors were transfected into ExpiCHO-S cells using the ExpiFectamine CHO Transfection kit (ThermoFisher Scientiﬁc Inc.) to express P38B antibody, which was further
puriﬁed using Protein G-Sepharose (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).
2.3. Antitumor activity of P38B
Female BALB/c nude mice (5-week-old) were purchased fromCharles River (Kanagawa, Japan) and used in experiments when they
were 7 weeks old. CHO/dPDPN and CHO-K1 cells (0.3 mL of
1.33 × 108 /mL in RPMI) were mixed with 0.5 mL of BD Matrigel
Matrix Growth Factor Reduced (BD Biosciences, San Jose, CA, USA). A
100-μL suspension (containing 5 × 106 cells) was injected subcutaneously into the left ﬂanks of nude mice. After day 1, 100 μg ofP38B or dog IgG (Jackson ImmunoResearch Inc., PA, USA) in 100 μL
PBS were injected into the peritoneal cavity of each mouse. Additional
antibodies were injected on day 8 and day 15. Canine mononuclearcells (5 x 105 cells), which were obtained from Hokkaido University,
were injected surrounding the tumors on day 1, day 8, and day 15. The
tumor diameter and tumor volume were determined as previously described [20]. The mice were euthanized 17 days after cell implantation.
All data were expressed as the mean ± SEM. Statistical analysis wasperformed using the Tukey–Kramer test. P < 0.05 was considered
statistically signiﬁcant.

Fig. 1. Antitumor activity of P38B against CHO/dPDPN and CHO-K1. (A)Tumor volume of CHO/dPDPN xenografts. CHO/ dPDPN cells were injectedsubcutaneously into female nude mice. The indicated antibodies (100 μg/day;5 mg/kg) were administered intraperitoneally on days 1, 8, and 15 after cell
inoculation. The tumor volume was measured at the indicated time points. The
values are presented as mean ± SEM. (B) CHO-K1 cells were injected subcutaneously into female nude mice. The indicated antibodies (100 μg/day;5 mg/kg) were administered intraperitoneally, and canine mononuclear cells
were injected around the tumors on day 1, day 8, and day 15. The tumor volume was measured at the indicated time points. The values are presented asmean ± SEM. An asterisk indicates statistical signiﬁcance (* P < 0.05, TukeyKramer's test). n.s.: not signiﬁcant.

3. Results and discussion
We have previously reported the development of the mouse mAb,
PMab-38 against dPDPN [10]. The strong reaction of PMab-38 speciﬁcally with canine SCCs [15] and melanomas [16] indicated that PMab38 possesses cancer-speciﬁcity. We have previously established cancerspeciﬁc mAbs against human PDPN, such as LpMab-2 [8] and LpMab23 [21]. These cancer-speciﬁc mAbs may prove advantageous for24

Biochemistry and Biophysics Reports 17 (2019) 23–26

Y. Kato et al.

Fig. 2. Evaluation of antitumor activity of P38B against CHO/dPDPN and CHO-K1. (A) CHO/dPDPN xenograft mice models on day 17. (B) CHO-K1 xenograft
mice models on day 17. (C) Resected tumors of CHO/dPDPN xenografts. (D) Resected tumors of CHO-K1 xenografts. (E) Tumor weight of CHO/dPDPN xenografts(day 17). (F) Tumor weight of CHO-K1 xenografts (day 17). (G) Body weight of CHO/dPDPN xenografts (day 17). (H) Body weight of CHO-K1 xenografts (day 17).
The values are presented as mean ± SEM. An asterisk indicates statistical signiﬁcance (* P < 0.05, Tukey–Kramer's test). n.s.: not signiﬁcant.

dPDPN and CHO-K1 xenografts are depicted in Fig. 2C and D, respectively. The tumor weight of mice in P38B-treated was signiﬁcantly
lower than in the control dog IgG group in CHO/dPDPN xenograftmodels (Fig. 2E) although there was no diﬀerence in CHO-K1 xenograftmodels (Fig. 2F). However, body weight was not signiﬁcantly diﬀerent

day 17 (Fig. 1A). Conversely, P38B did not reduce tumor developmentof CHO-K1 xenograft (Fig. 1B). This observation indicated that P38B is
speciﬁc against dPDPN.
CHO/dPDPN and CHO-K1 xenograft mice models on day 17 areshown in Fig. 2A and B, respectively. The resected tumors of CHO/25

Biochemistry and Biophysics Reports 17 (2019) 23–26

Y. Kato et al.

among the two groups in the CHO/dPDPN xenograft models (Fig. 2G)
and the CHO-K1 xenograft models (Fig. 2H).
In conclusion, P38B is applicable for antibody therapy against canine cancers expressing dPDPN. Further studies on antitumor activities
against endogenous dPDPN-expressing tumors are necessary to obtain amore detailed understanding of antibody therapy against canine cancers.

[7] E.P. Retzbach, S.A. Sheehan, E. Nevel, A. Batra, T. Phi, Y. Kato, S. Baredes,M. Fatahzadeh, A.J. Shienbaum, G.S. Goldberg, Podoplanin emerges as a functionally relevant oral cancer biomarker and therapeutic target, Oral Oncol. (2018).
[8] Y. Kato, M.K. Kaneko, A cancer-speciﬁc monoclonal antibody recognizes the aberrantly glycosylated podoplanin, Sci. Rep. 4 (2014) 5924.
[9] G. Zimmer, H.D. Klenk, G. Herrler, Identiﬁcation of a 40-kDa cell surface sialoglycoprotein with the characteristics of a major inﬂuenza C virus receptor in a
Madin-Darby canine kidney cell line, J. Biol. Chem. 270 (1995) 17815–17822.
[10] R. Honma, M.K. Kaneko, S. Ogasawara, Y. Fujii, S. Konnai, M. Takagi, Y. Kato,
Speciﬁc detection of dog podoplanin expressed in renal glomerulus by a novel
monoclonal antibody PMab-38 in immunohistochemistry, Monoclon. Antib.
Immunodiagn. Immunother. 35 (2016) 212–216.
[11] S. Yamada, S. Itai, M.K. Kaneko, Y. Kato, PMab-48 recognizes dog podoplanin oflymphatic endothelial cells, Monoclon. antibodies Immunodiagn. Immunother. 37(2018) 63–66.
[12] Y.W. Chang, S. Yamada, M.K. Kaneko, Y. Kato, Epitope mapping of monoclonal
antibody PMab-38 against dog podoplanin, Monoclon. Antibodies Immunodiagn.
Immunother. 36 (2017) 291–295.
[13] S. Yamada, M.K. Kaneko, S. Itai, Y.W. Chang, T. Nakamura, M. Yanaka,S. Ogasawara, T. Murata, H. Uchida, H. Tahara, H. Harada, Y. Kato, Epitope
mapping of monoclonal antibody PMab-48 against dog podoplanin, Monoclon.
Antibodies Immunodiagn. Immunother. 37 (2018) 162–165.
[14] M.K. Kaneko, R. Honma, S. Ogasawara, Y. Fujii, T. Nakamura, N. Saidoh, M. Takagi,Y. Kagawa, S. Konnai, Y. Kato, PMab-38 recognizes canine podoplanin of squamouscell Carcinomas, Monoclon. Antib. Immunodiagn. Immunother. 35 (2016)
263–266.
[15] S. Ogasawara, R. Honma, M.K. Kaneko, Y. Fujii, Y. Kagawa, S. Konnai, Y. Kato,
Podoplanin expression in Canine Melanoma, Monoclon. Antib. Immunodiagn.
Immunother. 35 (2016) 304–306.
[16] Y. Kato, T. Mizuno, S. Yamada, T. Nakamura, S. Itai, M. Yanaka, M. Sano,
M.K. Kaneko, Establishment of P38Bf, a core-fucose-deﬁcient mouse-canine chimeric antibody against dog podoplanin, Monoclon. Antib. Immunodiagn.
Immunother. 37 (2018) 218–223.
[17] Y. Fujii, M. Kaneko, M. Neyazaki, T. Nogi, Y. Kato, J. Takagi, PA tag: a versatile
protein tagging system using a super high aﬃnity antibody against a dodecapeptide
derived from human podoplanin, Protein Expr. Purif. 95 (2014) 240–247.
[18] Y. Fujii, M.K. Kaneko, S. Ogasawara, S. Yamada, M. Yanaka, T. Nakamura,N. Saidoh, K. Yoshida, R. Honma, Y. Kato, Development of RAP Tag, a novel tagging
system for protein detection and puriﬁcation, Monoclon. Antibodies Immunodiagn.
Immunother. 36 (2017) 68–71.
[19] Y. Fujii, M.K. Kaneko, Y. Kato, MAP tag: a novel tagging system for protein puriﬁcation and detection, Monoclon. Antibodies Immunodiagn. Immunother. 35(2016) 293–299.
[20] Y. Kato, A. Kunita, S. Abe, S. Ogasawara, Y. Fujii, H. Oki, M. Fukayama, Y. Nishioka,M.K. Kaneko, The chimeric antibody chLpMab-7 targeting human podoplaninsuppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing
activity, Oncotarget 6 (2015) 36003–36018.
[21] S. Yamada, M.K. Kaneko, Y. Kato, LpMab-23: a cancer-speciﬁc monoclonal antibody
against human Podoplanin, Monoclon. Antib. Immunodiagn. Immunother. 36(2017) 72–76.
[22] L.M. Bergeron, E.E. McCandless, S. Dunham, B. Dunkle, Y. Zhu, J. Shelly, S. Lightle,
A. Gonzales, G. Bainbridge, Comparative functional characterization of canine IgG
subclasses, Vet. Immunol. Immunopathol. 157 (2014) 31–41.
[23] S.M. Rue, B.P. Eckelman, J.A. Efe, K. Bloink, Q.L. Deveraux, D. Lowery, M. Nasoﬀ,
Identiﬁcation of a candidate therapeutic antibody for treatment of canine B-celllymphoma, Vet. Immunol. Immunopathol. 164 (2015) 148–159.

Acknowledgments
We thank Akiko Harakawa, Takuro Nakamura, Miyuki Yanaka,
Kayo Hisamatsu, Saori Handa, and Yoshimi Nakamura for their excellent technical assistance.
Funding
This research was supported in part by AMED under Grant nos.
JP18am0101078 (Y.K.), JP18am0301010 (Y.K.), and JP18ae0101028(Y.K.), and by JSPS KAKENHI Grant nos. 17K07299 (M.K.K.) and16K10748 (Y.K.).
Appendix A. Transparency document
Supplementary data associated with this article can be found in theonline version at doi:10.1016/j.bbrep.2018.11.005.
References[1] S. Breiteneder-Geleﬀ, K. Matsui, A. Soleiman, P. Meraner, H. Poczewski, R. Kalt,
G. Schaﬀner, D. Kerjaschki, Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis, Am. J. Pathol.
151 (1997) 1141–1152.
[2] N. Kimura, I. Kimura, Podoplanin as a marker for mesothelioma, Pathol. Int. 55(2005) 83–86.
[3] S. Abe, Y. Morita, M.K. Kaneko, M. Hanibuchi, Y. Tsujimoto, H. Goto, S. Kakiuchi,
Y. Aono, J. Huang, S. Sato, M. Kishuku, Y. Taniguchi, M. Azuma, K. Kawazoe,Y. Sekido, S. Yano, S. Akiyama, S. Sone, K. Minakuchi, Y. Kato, Y. Nishioka, A novel
targeting therapy of malignant mesothelioma using anti-podoplanin antibody, J.
Immunol. 190 (2013) 6239–6249.
[4] Y. Kato, M. Kaneko, M. Sata, N. Fujita, T. Tsuruo, M. Osawa, Enhanced expressionof Aggrus (T1alpha/podoplanin), a platelet-aggregation-inducing factor in lungsquamous cell carcinoma, Tumor Biol. 26 (2005) 195–200.
[5] Y. Kato, I. Sasagawa, M. Kaneko, M. Osawa, N. Fujita, T. Tsuruo, Aggrus: a diagnostic marker that distinguishes seminoma from embryonal carcinoma in testiculargerm cell tumors, Oncogene 23 (2004) 8552–8556.
[6] K. Mishima, Y. Kato, M.K. Kaneko, R. Nishikawa, T. Hirose, M. Matsutani, Increased
expression of podoplanin in malignant astrocytic tumors as a novel molecularmarker of malignant progression, Acta Neuropathol. 111 (2006) 483–488.

26