MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 5, 2018
Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0035

Establishment of P38Bf, a Core-Fucose-Deficient
Mouse-Canine Chimeric Antibody Against Dog Podoplanin
Yukinari Kato,1,2 Takuya Mizuno,3 Shinji Yamada,1 Takuro Nakamura,1 Shunsuke Itai,1Miyuki Yanaka,1 Masato Sano,1 and Mika K. Kaneko1

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed in normal tissues, including lymphatic
endothelial cells, pulmonary type I alveolar cells, and renal podocytes. The overexpression of PDPN in cancers is
associated with hematogenous metastasis by interactions with the C-type lectin-like receptor 2 (CLEC-2). We havepreviously reported the development of a mouse monoclonal antibody (mAb) clone, PMab-38 (IgG1, kappa), against
dog PDPN (dPDPN). PMab-38 reacted strongly with canine squamous cell carcinomas and melanomas, but not withlymphatic endothelial cells, indicating its cancer specificity. In this study, we developed and produced several mousecanine chimeric antibodies originating from PMab-38. A mouse-canine chimeric antibody of subclass A (P38A) and amouse-canine chimeric antibody of subclass B (P38B) were transiently produced using ExpiCHO-S cells. Corefucose-deficient P38B (P38Bf) was developed using FUT8 knockout ExpiCHO-S cells. We compared the binding
affinities, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) of
P38A, P38B, and P38Bf against Chinese hamster ovary (CHO)/dPDPN cells. Flow cytometry analysis showed that
the KD of P38A, P38B, and P38Bf were 1.9 · 10-7, 5.2 · 10-9, and 6.5 · 10-9, respectively. Both P38B and P38Bf
revealed high ADCC activities against CHO/dPDPN cells; P38Bf demonstrated significantly higher ADCC compared with P38B, especially at low concentrations. P38B and P38Bf exhibited higher CDC activities against CHO/
dPDPN cells. Conversely, P38A did not exhibit any ADCC or CDC activity. In summary, P38Bf is a good candidate
for antibody therapy against dPDPN-expressing canine cancers.
Keywords: mouse-canine chimeric antibody, dog podoplanin, dPDPN, monoclonal antibody

IntroductionP

odoplanin (PDPN) is known to be expressed in normal
tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, renal podocytes, chondrocytes,
myofibroblasts, and mesothelial cells.(1) An elevated expression of PDPN is also observed in different types oftumors, such as squamous cell carcinomas (SCCs),(2) testicular tumors,(3) glioblastoma,(4) and mesothelioma.(5,6) Recent
clinical studies have provided evidence for the association
between increased PDPN expression and poor disease prognosis,(7) indicating that the establishment of anti-PDPN
monoclonal antibodies (mAbs) is critical for developing
novel therapeutic strategies against cancer development andmetastatic progression.(8)Dog PDPN (dPDPN) was previously reported as gp40.(9)We developed two mAbs namely, PMab-38 (mouse IgG1,
kappa)(10) and PMab-48 (mouse IgG1, kappa),(11) which specifically recognize dPDPN. PMab-38 recognized dPDPN ofrenal epithelial cells, but did not react with lymphatic endothelial cells.(10) Conversely, PMab-48 reacted not only withrenal epithelial cells but also with lymphatic endothelial
cells.(11) Tyr67 and Glu68 of dPDPN were determined as thecritical epitopes of PMab-38.(12) Contrastingly, Asp29, Asp30,
Ile31, Ile32, and Pro33 of dPDPN were found to be necessary forrecognition of PMab-48.(13) Using immunohistochemistry, we
further demonstrated that PMab-38 reacted with 83% of canine
SCCs (15/18 cases)(14) and 90% of melanomas (9/10 cases),(15)indicating that PMab-38 is applicable for antibody-based therapy for canine cancers. In this study, we produced several

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.

2
3ª Yukinari Kato et al. 2018; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative
Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.

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MOUSE-CANINE CHIMERIC ANTI-DPDPN MAB

mouse-canine chimeric antibodies from PMab-38 and investigated their antibody-dependent cellular cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC) activities.
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 cell line was obtained
from the American Type Culture Collection (ATCC, Manassas, VA). In our previous studies, we inserted dPDPN with
an N-terminal PA tag and a C-terminal RAP tag-MAP tag(PA-dPDPN-RAP-MAP) in a pCAG-Ble vector (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan).(10) The
PA tag,(16) RAP tag,(17) and MAP tag(18) consist of 12 aminoacids each, namely, GVAMPGAEDDVV, DMVNPGLEDRIE, and GDGMVPPGIEDK, respectively. CHO-K1 cells

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were transfected with pCAG-Ble/PA-dPDPN-RAP-MAP
using Gene Pulser Xcell electroporation system (Bio-Rad
Laboratories, Inc., Berkeley, CA) resulting in the cell line
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 mg/mL of streptomycin, and 25 mg/mLof amphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified atmosphere of 5% CO2 and 95% air.
Antibodies

PMab-38, a mouse anti-dPDPN mAb (IgG1, kappa), wasdeveloped as previously described.(10) To generate a mousecanine (subclass A) chimeric antibody, P38A, the appropriate
VH and VL cDNAs of mouse PMab-38 and the CH and CL of

FIG. 1. Production of P38A (canine IgGA), P38B (canine IgGB), and P38Bf (canine IgGB; core-fucose deficient) from
PMab-38 (mouse IgG1).

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KATO ET AL.

FIG. 2. Flow cytometry. CHO/dPDPN cells were treated with (A) P38A, (B) P38B, and (C) P38Bf, followed by treatment
with FITC-conjugated anti-canine IgG. Fluorescence data were collected using a cell analyzer. CHO, Chinese hamster
ovary; dPDPN, dog podoplanin.
canine IgG subclass A were subcloned into pCAG-Ble andpCAG-Neo vectors (FUJIFILM Wako Pure Chemical Corporation), respectively. Similarly, to generate a mouse-canine(subclass B) chimeric antibody, P38B, the appropriate VH and
VL cDNAs of mouse PMab-38 and the CH and CL of canine
IgG subclass B were subcloned into pCAG-Ble and pCAGNeo vectors (FUJIFILM Wako Pure Chemical Corporation),
respectively. To express P38A and P38B, antibody expression
vectors were transfected into ExpiCHO-S cells using the ExpiFectamine CHO Transfection kit (Thermo Fisher Scientific,Inc.). To generate P38Bf, antibody expression vectors weretransfected into BINDS-09 (FUT8-knocked out ExpiCHO-S
cells*) using the ExpiFectamine CHO Transfection kit. P38A,
P38B, and P38Bf were purified using Protein G-Sepharose(GE Healthcare Bio-Sciences, Pittsburgh, PA).

After washing with 0.1% bovine serum albumin in phosphatebuffered saline, the cells were treated with P38A, P38B, andP38Bf (0.1–10 mg/mL) for 30 minutes at 4C, followed bytreatment with FITC-conjugated anti-dog IgG (1:200; SigmaAldrich Corp., St. Louis, MO). Fluorescence data were acquired
using the Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).
Determination of binding affinity using flow cytometry

CHO/dPDPN cells (2 · 105) were resuspended in 90 mL ofserially diluted P38A, P38B, and P38Bf (6 ng/mL to 100 mg/mL), followed by the addition of secondary anti-dog IgG(1:30; Sigma-Aldrich Corp., St. Louis, MO). Fluorescencedata were collected using a cell analyzer (EC800). KD was
obtained by fitting the binding isotherms using the built-in
one-site binding models in GraphPad PRISM 6 (GraphPad
Software, La Jolla, CA).

Flow cytometry

Cells were harvested after brief exposure to 0.25% trypsin/
1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.).
*www.med-tohoku-antibody.com/topics/001_paper_cell.htm

Antibody-dependent cellular cytotoxicity

Canine lymphokine-activated killer (LAK) cells, used as
effector cells, were prepared from peripheral blood mononuclear cells from a healthy beagle dog by culturing in theMOUSE-CANINE CHIMERIC ANTI-DPDPN MAB

presence of 1000 IU/mL of human recombinant IL-2 for 1
week. CHO-K1/luc and CHO/dPDPN/luc cells were used astarget cells.(19) Both cells were generated by transducing theluciferase reporter vector into CHO-K1 and CHO/dPDPN
cells. CHO-K1/luc cells and CHO/dPDPN/luc cells (5 · 103)
were seeded into 96-well flat-bottom microtiter plates containing dog IgG whole molecule ( Jackson ImmunoResearch,
Inc., PA), P38A, P38B, or P38Bf. After incubating on ice for
15 minutes, the LAK cells were added at an effector/target
ratio of 20:1. For maximal killing control, 2% Triton X-100
was added. Cultures were incubated further for 4 hours followed by cell lysis using the ONE-Glo Luciferase assay
system (Promega, Madison, WI) according to manufacturer’s
instructions. Luciferase activities were detected by the
ARVO X4 system (PerkinElmer, Waltham, MA). ADCC was
calculated as follows: duplicate wells were averaged andpercent lysis was calculated from the data using the following
equation: % specific lysis = 100 · (spontaneous death RLU test RLU)/(spontaneous death RLU - maximal killing RLU).
Experiments were repeated thrice and results are shown as amean of three independent experiments.

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human IgG1 and IgG3, respectively.(21) Furthermore, thesubclasses B and C can induce CDC. Rue et al. established ananti-canine CD20 mAb (1E4) to treat canine B-cell lymphoma and produced mouse-canine chimeric antibodies.(22)
They also showed that 1E4-cIgGB (subclass B) and 1E4cIgGC (subclass C) led to significant depletion of B-celllevels in healthy beagle dogs.
In this study, we first developed several mouse-canine
chimeric antibodies by combining the variable regions ofPMab-38 with the constant regions of different canine IgG
subclasses (Fig. 1). A canine IgG subclass A corresponding to
the human IgG2 does not possess ADCC and CDC activities.(21) In contrast, a canine IgG subclass B corresponding
to human IgG1 possesses high ADCC and CDC activities.
Furthermore, deletion of core-fucose from the human IgG1

Complement-dependent cytotoxicity

Luciferase-expressing CHO-K1 cells and CHO/dPDPN
cells (5 · 103) were seeded into 96-well round-bottom microtiter plates containing either whole molecule dog IgG,
P38A, P38B, or P38Bf. After incubating on ice for 20 minutes, LOW-TOX-H rabbit complement at a dilution of 1:40
(CEDALANE, Ontario, Canada) was added to each well andincubated further for 90 minutes. This was followed by cell
lysis using the ONE-Glo Luciferase assay system (Promega)according to the manufacturer’s instructions. Luciferase activities were detected by the ARVO X4 system (PerkinElmer). CDC was calculated as follows: duplicate wells
were averaged and percent lysis was calculated from the datausing the following equation: % specific lysis = 100 · (spontaneous death RLU - test RLU)/(spontaneous death RLU maximal killing RLU). The experiments were repeated thrice
and results are shown as an average of three independent
experiments.
Results and Discussion

Previously, we developed a mouse anti-dPDPN mAb,
namely, PMab-38, by immunization using dPDPN proteins,which were expressed in CHO-K1 cells.(10) PMab-38 exhibits ahigh sensitivity and specificity against dPDPN and is highly
suitable for the detection of PDPN-expressing cancer cells of
SCCs and melanomas in immunohistochemical analyses.(14,15)
Conversely, PMab-38 reacts weakly with podocytes of the canine kidney and does not recognize lymphatic endothelial cells,
indicating that PMab-38 is a cancer-specific mAb. We previously established cancer-specific mAbs (CasMabs) against human PDPN, such as LpMab-2(8) and LpMab-23.(20) Thesecancer-specific mAbs may prove advantageous for targetingcancer cells without adverse effects.
Few studies have already been performed concerning the
subclasses (A, B, C, and D) of canine IgGs.(21,22) Bergeron
et al. clearly showed that the canine subclasses A and D
appear effector-function negative, while the subclasses B andC bind canine Fc gamma receptors and are positive for
antibody-dependent cellular cytotoxicity (ADCC) similar to

FIG. 3. Determination of binding affinities using flow
cytometry. CHO/dPDPN cells were suspended in 90 mL ofserially diluted antibodies (6 ng/mL to 100 mg/mL): (A)P38A, (B) P38B, and (C) P38Bf, followed by treatment with
FITC-conjugated anti-canine IgG. Fluorescence data werecollected using a cell analyzer. GeoMean, geometric mean.

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FIG. 4. ADCC activity of P38A, P38B, and P38Bf against
dPDPN-expressing CHO cells. ADCC was determined byluciferase-based cytotoxicity assay. CHO/dPDPN/luc cells wereincubated with serially diluted dog IgG, P38A, P38B, and P38Bf
antibodies (0.001–10 mg/mL) in the presence of LAK cells at aneffector: target ratio of 20:1 for 4 hours. Luciferase activity wasmeasured by lysis of live cells after appropriate incubation time
periods. Values presented are mean – SEM of three independent experiments. *P < 0.05, **P < 0.01, Tukey–Kramer’s test.
ADCC, antibody-dependent cellular cytotoxicity; LAK,
lymphokine-activated killer; SEM, standard error of the mean.
has been reported to induce significantly higher ADCC activity.(23) Therefore, we produced a core-fucose-deficientmouse-canine chimeric antibody using FUT8 knockout celllines. Resultantly, P38A (subclass A), P38B (subclass B), andP38Bf (core-fucose-deficient subclass B) were developed. As
shown in Figure 2, three chimeric antibodies showed highsensitivity against CHO/dPDPN cells. Among these, P38B
and P38Bf showed a significantly higher reaction comparedwith P38A at low concentrations suggesting that (i) the
constant regions of the subclass B might be more stable than

KATO ET AL.

those of subclass A in this study and (ii) the depletion of corefucose from canine IgG might not reduce sensitivity of theantibody. We next compared the binding affinity of P38A,
P38B, and P38Bf and the KD of P38A, P38B, and P38Bf wasdetermined to be 1.9 · 10-7, 5.2 · 10-9, and 6.5 · 10-9, respectively, using flow cytometry analysis (Fig. 3). This
clearly indicated a higher binding affinity of P38B and P38Bf
compared with P38A. In this study, we utilized the constantregions of canine IgGs, which were obtained commercially.
We aim to further investigate the binding affinities of canine
IgGs possessing different constant regions obtained fromother sources, to clearly discuss the binding affinities andstability of various canine IgGs.
We next investigated ADCC activities using P38A,
P38B, and P38Bf. To this end, we used CHO/dPDPN/luc astarget cells and LAK cells as effector cells. Both P38B and
P38Bf revealed high ADCC activities against CHO/
dPDPN/luc cells; P38Bf demonstrated significantly higher
ADCC than P38B, especially at low concentrations such as
1 ng/mL (Fig. 4). Both P38B and P38Bf did not exhibit
ADCC against parental CHO-K1/luc cells (Supplementary
Fig. S1), indicating that cytotoxicity is dependent on
dPDPN overexpressed in CHO-K1 cells. Conversely,
P38A did not exhibit ADCC activity against CHO/dPDPN/
luc cells (Fig. 4), confirming that the subclass A of canine
IgG could not induce ADCC.
Furthermore, we investigated CDC activities using P38A,
P38B, and P38Bf by using CHO/dPDPN/luc as target cells.
As shown in Figure 5, P38B and P38Bf showed high CDC
activities against CHO/dPDPN cells. Both P38B and P38Bf
did not show CDC against parental CHO-K1/luc cells (Supplementary Fig. S2), indicating that P38B- and P38Bfmediated CDC are dependent on dPDPN overexpression in
CHO-K1 cells. Contrastingly, P38A did not exhibit any CDC
activity (Fig. 5), which confirmed that the subclass A of canine IgG could not induce CDC.
Taken together, P38Bf is applicable for antibody therapy
against canine cancers expressing dPDPN. Further studies on
ADCC and CDC are necessary to obtain a more detailed
understanding of endogenous dPDPN-expressing canine
cancer cells.
Acknowledgments

We thank Kayo Hisamatsu, Saori Handa, and Yoshimi
Nakamura for their excellent technical assistance. This
research was supported, in part, by AMED under grant
numbers JP18am0301010 (Y.K.), JP18am0101078 (Y.K.),
and JP18ae0101028 (Y.K.), and by JSPS KAKENHI grant
number 17K07299 ( M.K.K.) and grant number 16K10748(Y.K.).
Author Disclosure Statement

FIG. 5. CDC activity of P38A, P38B, and P38Bf against
dPDPN-expressing CHO cells. CDC was determined byluciferase-based cytotoxicity assay. CHO/dPDPN/luc cells
were incubated with a rabbit complement at a dilution of
1:40 in the presence of serially diluted dog IgG, P38A,
P38B, and P38f antibodies (0.156–10 mg/mL) for 90 minutes
in 96-well plates. Luciferase activity was measured after lysis
of live cells. Values presented are mean – SEM of three independent experiments. **P < 0.01, Tukey–Kramer’s test. CDC,
complement-dependent cytotoxicity; n.s., not significant.

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
JapanE-mail: yukinarikato@med.tohoku.ac.jp;
yukinari-k@bea.hi-ho.ne.jp
Received: September 14, 2018
Accepted: September 29, 2018