MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 2, 2020
Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0001

Original Articles

Antibody–Drug Conjugates Using Mouse–Canine Chimeric
Anti-Dog Podoplanin Antibody Exerts Antitumor
Activity in a Mouse Xenograft Model
Yukinari Kato,1,2 Yuji Ito,3 Tomokazu Ohishi,4 Manabu Kawada,4 Takuro Nakamura,1Yusuke Sayama,1 Masato Sano,1 Teizo Asano,1 Miyuki Yanaka,1 Saki Okamoto,1
Saori Handa,1 Yu Komatsu,1 Junko Takei,1 and Mika K. Kaneko1

Antibody–drug conjugates (ADCs), which consist of a monoclonal antibody (mAb), a linker, and a payload, can delivera drug to cancer tissues. We previously produced an anti-dog podoplanin (dPDPN) mAb, PMab-38, which reacts withdPDPN-expressing canine melanomas and squamous cell carcinomas (SCCs), but not with dPDPN-expressing canine
type I alveolar cells or lymphatic endothelial cells, indicating that PMab-38 possesses cancer specificity. In this study,
we developed an ADC, P38B-DM1, using the mouse–canine chimeric anti-dPDPN antibody, P38B as the antibody,a peptide linker, and emtansine as the payload using the chemical conjugation by affinity peptide (CCAP) method.
We investigated its cytotoxicity against dPDPN-overexpressed Chinese hamster ovary (CHO/dPDPN) cells in vitro
and its antitumor activity using a mouse xenograft model of CHO/dPDPN cells. P38B-DM1 showed cytotoxicity to
CHO/dPDPN cells in a dose-dependent manner in vitro. Furthermore, P38B-DM1 exhibited higher antitumor activity
than P38B in the mouse xenograft model. These results suggest that P38B-DM1, developed using the CCAP method,is useful for antibody therapy against dPDPN-expressing canine SCCs and melanomas.
Keywords: antibody–drug conjugate, dog podoplanin, monoclonal antibody

antibody Fc fragment as a novel site-specific conjugation
system.(3) Utilizing CCAP, various kinds of therapeutic molecules, from low molecular weight compounds to proteins, canbe attached to the antibody Fc through the affinity peptide. In
this study, we investigated an ADC consisting of a conjugate
between antibody and human serum albumin (HSA)-modified
with emtansine (DM1; a tubulin polymerization inhibitor).
Podoplanin (PDPN), which is also known as Aggrus,
T1alpha, PA2.26, OTS-8, and gp36, has been reported to be
expressed in normal tissues, including podocytes of the kidney, lymphatic endothelial cells, and type I alveolar cells of
the lung.(4) PDPN is also overexpressed in many cancers,
such as glioblastomas,(5) lung cancers,(6) and mesotheliomas.(7,8) Several clinical studies have investigated the association between PDPN overexpression and poor diseaseprognosis,(9) suggesting that anti-PDPN mAbs could be used
for novel therapeutic strategies against cancer development
and metastatic progression.(10)Introduction

A

ntibody–drug conjugates (ADCs), which consistof an antibody, a linker, and a payload, possess theability to deliver a drug to specific cells. Several sitespecific chemical conjugation strategies with native monoclonal antibodies (mAbs) have been developed, including
residue-selective labeling, disulfide rebridging, and affinitypeptide-mediated site-specific chemical conjugation technologies.(1) The most popular antibody conjugation system
is an amine coupling, using the activated carboxylate moiety, which has been used to generate a typical ADC, T-DM1(Kadcyla), used against breast cancers. However, to overcome difficulties involved in controlling the modificationsite and the drug concentration (drug-to-antibody ratio[DAR]), site-specific modification of the antibody has beenfavored.(2) Recently, chemical conjugation by affinity peptide (CCAP) has been developed using affinity peptide to the1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
3
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, Kagoshima University, Kagoshima, Japan.
4
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Shizuoka, Japan.
2ª Yukinari Kato et al. 2020; 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.

37

38

Previously, we established an anti-dog podoplanin(dPDPN), PMab-38 mAb.(11) PMab-38 did not react withlymphatic endothelial cells, and recognized dPDPN of renal
epithelial cells weakly(11) although dPDPN is expressed inthose cells.(12) Immunohistochemical analyses demonstrated
that PMab-38 stained canine squamous cell carcinomas(13) andcanine melanomas,(14) indicating that PMab-38 demonstrated
cancer specificity. Epitope mapping studies revealed Tyr67
and Glu68 of dPDPN to be the critical epitopes of PMab-38.(15)
Furthermore, we produced a mouse–canine chimeric antidPDPN antibody, P38B, derived from PMab-38.(16) P38B
possesses antibody-dependent cellular cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC) activities
against Chinese hamster ovary (CHO)/dPDPN cells. Although
P38B exhibited antitumor activity in mouse xenograft models
using CHO/dPDPN,(17) that activity was insufficient for targeting canine cancers. In this study, we investigated whether
an ADC of P38B (P38B-DM1) showed superior antitumor
activity in a CHO/dPDPN xenograft model.
Materials and Methods
Antibodies

PMab-38, a mouse anti-dPDPN mAb was developed in ourprevious study.(11) To develop the mouse–canine chimericantibody P38B, VH of PMab-38 and CH of canine immunoglobulin G (IgG) subclass B were subcloned into the pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan), and VL of PMab-38 and CL of canine IgG were subcloned into the pCAG-Neo vector (FUJIFILM Wako Pure
Chemical Corporation).(16) Using the ExpiFectamine CHO
Transfection kit (Thermo Fisher Scientific, Inc., Waltham,MA), the expression vectors were transfected into ExpiCHO-S
cells to express P38B antibody. P38B was purified using Protein G-Sepharose (GE Healthcare Biosciences, Pittsburgh, PA).
Preparation of ADC P38B-DM1

For the preparation of the ADC using P38B, azide-modified
CCAP peptide (FNMQQQ(Lys[Azide])RFYEALHDPNLNE
EQRNARI(Lys[Azide])SIRDDPSRR(Lys[Azide])RR(Lys[Azide])RR(Lys[Azide])-CONH2), where Lys[Azide] is
5-azide-2-amino pentanoic acid) derived from Z34C peptide(18) was used. This CCAP peptide was N-terminally modified with disuccinimidyl glutarate to give the CCAP reagent(N-terminal succinimidyl glutaryl peptide). P38B (4.5 mL,
5.1 mg/mL; 34 mM) in phosphate-buffered saline (PBS) was
mixed with 22 mL of 27.9 mM CCAP reagent dissolved in dimethyl sulfoxide and incubated for 1 hour at room temperature.
The reaction of antibody with CCAP peptide generated themonovalent and divalent antibodies owing to the two modified sites of CCAP peptide on Fc. To remove the unmodified
and the divalent modified antibodies, the reaction mixture
was subjected to affinity chromatography conjugated with
Z33 affinity peptide (Acetyl-FNMQQQRRFYEALHDPNLNEEQRNARIRSIRDDPSGSGSK-NH2). The monovalentmodified antibody was recovered by eluting with mild acidic
buffer. Based on ion-exchange chromatography, the purity ofmonovalent modified antibody was estimated to be 95%,
leaving the residual contaminant of 5% unmodified antibody.
The modification of HSA with DM-1 was performed asfollows. HSA (20 mg) dissolved in 1.35 mL of 50 mM HEPES

KATO ET AL.

(pH 7.0) was reduced with 0.5 mM dithiothreitol (DTT). The
partially reduced HSA was supplied to a Hi Trap Capto Q
column (GE Healthcare Biosciences) equilibrated with
50 mM HEPES buffer (pH 7.0), washed with the same buffer,and eluted with buffer containing 1 M NaCl. The eluted HSA(50 mM) was reacted with 250 mM dibenzocyclooctyne(DBCO)-PEG4-Maleimide (Click Chemistry Tools) and incubated for 2 hours at a room temperature. The DBCOmodified HSA formed (7 mM) was subsequently reacted witha 20-fold excess of DM1-SMCC (MedChemExpress) for 12hours at 37C.
After the reaction, the sample was supplied to a Hi Trap
Capto Q column (GE Healthcare Biosciences) equilibrated
with 50 mM HEPES buffer (pH 7.0), washed with the same
buffer, and eluted with buffer containing 1 M NaCl. The drug
(DM-1) attachment efficiency to the HSA (DAR) was estimated to be 2.9 using matrix-associated laser desorption/ionization time-of-flight mass spectroscopy (autoflex
speed TOF; Bruker Daltonics).
Finally, azide-modified Z33-monovalent-modified antibody (10 mM) and DM-1/DBCO-modified HSA (20 mM)
were reacted at pH 7.0 for 12 hours at room temperature
through the click chemistry. The generation of antibody-HSA
conjugate was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The reaction
mixture was subjected to a Z33 affinity peptide column toremove the excess HSA eluted in the excluded fraction. The
bound HSA-monovalent modified antibody was eluted by
0.1 M acetic acid buffer (pH 4.0) and dialyzed in PBS for use.
Cell lines and animals

We previously inserted dPDPN with an N-terminal PA
tag (GVAMPGAEDDVV)(19) and a C-terminal RAP tag(DMVNPGLEDRIE)(20)-MAP tag (GDGMVPPGIEDK)(21)(PA-dPDPN-RAP-MAP) in a pCAG-Ble vector (FUJIFILM
Wako Pure Chemical Corporation).(11) CHO-K1 cells(American Type Culture Collection, Manassas, VA) weretransfected with pCAG-Ble/PA-dPDPN-RAP-MAP using
Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Inc., Berkeley, CA) for developing CHO/dPDPN.
CHO/dPDPN and CHO-K1 cells were cultured using
RPMI-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan)
supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc.). Antibiotics, such as 100 U/mL
penicillin, 100 mg/mL streptomycin, and 25 mg/mL amphotericin B (Nacalai Tesque, Inc.) were added into the medium.
Cells were cultivated at 37C in a humidified atmosphere of 5%
CO2 and 95% air.
Flow cytometry

Using 0.25% trypsin/1 mM ethylenediaminetetraacetic acid
(Nacalai Tesque, Inc.), CHO/dPDPN cells were harvested.
Cells were treated with P38B (0.01–10 mg/mL), P38B-DM1(0.01–10 mg/mL), or normal canine IgG (10 mg/mL; Jackson
ImmunoResearch, Inc., PA) for 30 minutes at 4C, followed bytreatment with fluorescein isothiocyanate-conjugated anticanine IgG (1:200; Thermo Fisher Scientific, Inc.). In all steps,
the cells were washed with 1% bovine serum albumin in PBS.
Fluorescence data were detected using the Cell Analyzer
EC800 (Sony Corp., Tokyo, Japan). We repeated these experiments twice.

ADC OF ANTI-DPDPN MAB
Cytotoxicity assay

Cytotoxicity assay by cell viability assessment was conducted using Cell Cloning Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). We prepared 96-well microplates by plating

39

CHO/dPDPN and CHO-K1 at 6000 cells/200 mL/well. The
plates were incubated for 24 hours at 37C in the presence of
5% CO2, then serially diluted antibodies were added in a
volume of 20 mL and cultured for 24–72 hours. After that, theculture medium was replaced with 100 mL of fresh medium

FIG. 1. Preparation of ADC P38B-DM1 using CCAP method. (A) The reaction of antibody with CCAP peptide generated
the monovalent and divalent antibodies owing to the two modified sites of CCAP peptide on Fc. To remove the unmodified
and the divalent-modified antibodies, the reaction mixture was subjected to to affinity chromatography conjugated with Z33
affinity peptide. (B) The modification of HSA with DM-1 was performed. (C) Sodium dodecyl sulfate–polyacrylamide gelelectrophoresis of conjugate products under reducing conditions. Lane 1: molecular weight marker; Lane 2: Z33-azide
monovalent modified P38B; Lane 3: Z33-azide monovalent modified P38B + DBCO-HSA-DM-1; Lane 4: Z33-azide
monovalent modified P38Bf (core fucose-deficient version of P38B); Lane 5: Z33-azide monovalent-modified P38Bf +DBCO-HSA-DM-1; Lane 6: DBCO-HSA-DM-1. H: heavy chain, L: light chain. DBCO, dibenzocyclooctyne; ADC,
antibody–drug conjugate; CCAP, chemical conjugation by affinity peptide; HSA, human serum albumin.

40with 10 mL CCK-8 solution. After a further 5- to 6-hour incubation in a CO2 incubator, the absorbance at 450 nm wasmeasured.
Antitumor activity

Female BALB/c nude mice (5 weeks old) were purchasedfrom Charles River (Kanagawa, Japan) and used in experiments when they were 6 weeks old. All animal experiments
were reviewed and approved by the Institutional Animal Careand Use Committee of Institute of Microbial Chemistry (Approval number: 2019-043) and were performed in accordancewith relevant guidelines and regulations to minimize animal
suffering.
CHO/dPDPN cells (0.3 mL at 1.33 · 108/mL in RPMI) and
0.5 mL of BD Matrigel Matrix Growth Factor Reduced(BD Biosciences, San Jose, CA) were mixed, and 5 · 106 cells
in 100 mL were injected into the left flank of nude mice subcutaneously. One day after cell inoculation, 100 mg of antibodies (P38B-DM1, P38B, or normal canine IgG) in 100 mL
of PBS were injected into the peritoneal cavity of each mouse.
On days 8 and 14, additional 100 mg of antibodies were in-

KATO ET AL.

jected into each mouse. As previously described, the tumor
diameter and tumor volume were determined.(22) The mice
were killed 17 days after cell implantation.
All data were expressed as mean – standard error of mean.
Statistical analysis was performed using the Tukey–Kramer
test, with p < 0.05 considered statistically significant.
Results

In this study, we developed an ADC, P38B-DM1, using themouse–canine chimeric anti-dPDPN antibody, P38B as theantibody, a peptide linker, and emtansine as the payload using the CCAP method as given in Figure 1A and B. SDSPAGE of azide-modified Z33-monovalent modified antibody
and its conjugate with DBCO-HSA-DM-1 (P38B-ADC) are
given in Figure 1C.
Antibodies are affinity proteins and their ability to bind thetarget molecule with high sensitivity and specificity is crucial
to their function. With this in mind, we examined the characteristics of P38B and P38B-DM1. Flow cytometry results(Fig. 2) showed that both P38B and P38B-DM1 exhibited
similar reactions with CHO/dPDPN cells in a dose-dependent

FIG. 2. Flow cytometry. CHO/dPDPN cells were treated with P38B (blue line; 10 or 1 mg/mL), P38B-DM1 (red line; 10or 1 mg/mL), and normal canine IgG (black line; 10 mg/mL), followed by treatment with fluorescein isothiocyanateconjugated anti-canine IgG. Fluorescence data were collected using a cell analyzer. IgG, immunoglobulin G; CHO, Chinese
hamster ovary; PDPN, dog podoplanin.

ADC OF ANTI-DPDPN MAB

manner. Furthermore, P38B-DM1 did not react with CHOK1 (Fig. 2), indicating that it was sensitive and specificagainst dPDPN.
Next, we investigated the cytotoxicity of P38B and P38BDM1 against CHO/dPDPN cells. P38B-DM1 demonstrated highcytotoxicity in a dose-dependent manner against CHO/dPDPN
cells compared with P38B ( p < 0.01, using Tukey–Kramer test
in more than 0.0625 mg/mL) and normal canine IgG ( p < 0.01,
using Tukey–Kramer test in more than 0.0625 mg/mL) (Fig. 3A).
In our previous study, P38B showed high ADCC in caninemononuclear cells, and showed CDC using rabbit complementin CHO/dPDPN.(16) In this study, in contrast, the P38B-DM1
showed high cytotoxicity against CHO/dPDPN cells withoutmononuclear cell complement, indicating that it was internalized
into CHO/dPDPN cells where DM1 exerted its cytotoxicity. We
also performed additional experiments to confirm that P38BDM1 did not show any toxicity against CHO-K1 cells. As given
in Figure 3B, P38B-DM1 did not show any toxicity against
CHO-K1 cells, whereas it killed CHO/dPDPN cells.
We further investigated antitumor activities of P38B-DM1
in vivo. CHO/dPDPN cells were subcutaneously implanted into
the flanks of nude mice. P38B, P38B-DM1, and control (dog
IgG) were injected three times (on days 1, 8 and 14 after cell
injection) into the peritoneal cavity of the mice. Tumor
formation was clearly observed in mice from the control,
P38B-treated, and P38B-DM1-treated groups in CHO/dPDPN
xenograft model.
We measured the tumor size in all the xenograft models andfound that P38B-DM1 significantly reduced tumor developmentof the CHO/dPDPN xenograft compared with control (dog IgG)
on days 8, 10, 14, and 17 (Fig. 4). However, the tumor volume
in the P38B-DM1-treated groups was not significantly reduced
compared with that in the P38B-treated groups because P38B
also significantly reduced tumor development compared with
control (dog IgG) on all days (Fig. 4). As reported in our previous study, P38B showed very high CDC activity against
CHO/hPDPN cells,(16) indicating that mouse complement of
nude mice might capacitate CDC to P38B.
We next measured the weight of resected tumors from all
xenograft models on day 17. The tumor weight of mice in
P38B-DM1-treated groups was significantly lower than that in
the control (dog IgG) group in CHO/dPDPN xenograft models(Fig. 5A). In contrast, the tumor weight of mice in the P38Btreated groups was not significantly reduced compared withthe control (Fig. 5A), indicating that P38B-DM1 exerted
stronger antitumor activity than P38B. Images of the resected
tumors of the CHO/dPDPN xenografts are given in Figure 5B.
There were no differences in mouse body weight between thethree groups (Fig. 6A), indicating that the P38B-DM1 was nottoxic to the mice. The appearance of the mice on day 17 isgiven in Figure 6B. Because P38B-DM1 could not bind to Fc
receptor of mouse mononuclear cells such as NK cells ormacrophages, ADCC-mediated killing might not occur in the
xenograft models. Although we could not exactly demonstrate
the mechanism of anti-tumor activities by P38B-DM1 in thisstudy, P38B-DM1 could be internalized into cells, and exerted
cytotoxicity.
Discussion

Cancer is a deadly disease that affects all eukaryotes. Almost all cancers found in humans are also observed in dogs,

41

FIG. 3. Cytotoxicity assay. (A) Cytotoxicity assay by cell
viability assessment was conducted using CCK-8 We prepared
96-well microplates by plating CHO/dPDPN cells at 6000
cells/200 mL/well. The plates were incubated for 24 hours at
37C in the presence of 5% CO2, then serially diluted antibodies were added in a volume of 20 mL and cultured for 24hours. After that, the culture medium was replaced with
100 mL of fresh medium with 10 mL CCK-8 solution. After afurther 5-hour incubation in a CO2 incubator, the absorbance at
450 nm was measured. The values are presented as mean –SEM. Asterisks indicate statistical significance (**p < 0.01,
Tukey–Kramer test). (B) Cytotoxicity assay by cell viability
assessment was conducted using CCK-8 We prepared 96-well
microplates by plating CHO/dPDPN and CHO-K1 cells at
6000 cells/200 mL/well. The plates were incubated for 24 hours
at 37C in the presence of 5% CO2, then serially diluted antibodies were added in a volume of 20 mL and cultured for
72 hours. After that, the culture medium was replaced with
100 mL of fresh medium with 10 mL CCK-8 solution. After afurther 6-hour incubation in a CO2 incubator, the absorbance at
450 nm was measured. Values are presented as mean – SEM.
Asterisks indicate statistical significance (**p < 0.01, Tukey–
Kramer test). n.s., not significant; CCK-8, Cell Cloning Kit-8;
SEM, standard error of mean.
suggesting that the treatment approaches used in humans may
also be effective in canine cancers. Antibody therapies havebeen shown to improve disease outcome in cancer patientsand ADCs are thought to be one of the most potent nextgeneration antibody therapies.(23)
We previously produced P38B, a mouse–canine chimericanti-dPDPN antibody of canine subclass B(16) because canine

42

KATO ET AL.

FIG. 4. Antitumor activity of P38B and P38B-DM1
against CHO/dPDPN. Tumor volume of CHO/dPDPN xenografts. CHO/dPDPN cells were injected subcutaneously
into female nude mice. The indicated antibodies (100 mg/day;
5 mg/kg) were administered intraperitoneally on days 1, 8,
and 14 after cell inoculation. The tumor volume was measured at the indicated time points. The values are presented
as mean – SEM. Asterisks indicate statistical significance(*p < 0.05, **p < 0.01, Tukey–Kramer test).

FIG. 6. Evaluation of the safety of P38B and P38B-DM1
against CHO/dPDPN. (A) Body weight of CHO/dPDPN
xenografts. (B) Images of CHO/dPDPN xenograft model
mice on day 17.

FIG. 5. Evaluation of antitumor activity of P38B and
P38B-DM1 against CHO/dPDPN. (A) Tumor weight of
CHO/dPDPN xenografts (day 17). (B) Images of resected
tumors of CHO/dPDPN xenografts. Values are presented asmean – SEM. The asterisk indicates statistical significance(*p < 0.05, Tukey–Kramer test). n.s., not significant.

subclass B possesses ADCC and CDC activities analogous tothe human IgG1 subclass.(24) We clearly demonstrated that
P38B possesses ADCC and CDC against CHO/dPDPN cells.(16)P38B reduced tumor development of CHO/dPDPN xenograftsignificantly compared with control dog IgG,(17) indicating that
P38B exerted antitumor activity against dPDPN-expressing tumors using ADCC and CDC activities. In those previous studies,
canine mononuclear cells were also injected into xenograftmodels to induce ADCC activity of P38B. In contrast, antitumor
activities of P38B were also observed in this study (Fig. 4), although canine mononuclear cells were not injected into the
xenograft models, indicating that CDC of P38B alone could
exert antitumor activity. However, the tumor weight of mice in
the P38B-treated groups was not reduced significantly compared with the control (dog IgG) group in CHO/dPDPN xenograft models (Fig. 4A), suggesting that CDC of P38B alone did
not exhibit sufficient antitumor activity.
In this study, an ADC, P38B-DM1 was generated by conjugating P38B and HSA-attached DM1 (Fig. 1). This ADC
showed almost the same binding ability to dPDPN as unmodified P38B (Fig. 2), indicating that the HSA modificationof the antibody did not affect antigen binding. Furthermore,
its efficacy, as measured by its tumor regression activity, wasconfirmed by in vitro (Fig. 3) and in vivo (Figs. 4 and 5).
P38B + DBCO-HSA-DM-1 conjugates will be intracellularly

ADC OF ANTI-DPDPN MAB

incorporated by endocytosis through the binding with dPDPN.
The incorporated conjugate is degraded by proteases in lysosome to generate the DM1-attached Lys amino acid oroligopeptide, which can work as a killing agent through tubulin polymerization inhibition to induce apoptosis. The IC50
of P38B-DM1 was estimated to be 0.048 mg/mL in our preliminary experiment, which corresponds to 238 pM, assuming
the molecular weight of P38B-DM1 is *210,000. This value
is larger than the IC50 of T-DM1 (50 pM) against the HER2 +SK-BR3 breast cancer cell line despite the similar DAR for
both (2.9 for P38B-DM1 and 3.5 for T-DM1). However, thedifference in IC50 may be arise from a difference in the expression levels of the target antigens between the cell types.
A detailed comparison in the near future is advised to elucidate the advantages of these ADCs.
In this study, we examined the effects of P38B, a mouse–canine chimeric anti-dPDPN antibody of subclass B and its
ADC with emtansine (P38B-DM1) on CHO/dPDPN cells.
Results indicate a significant increase in the cytotoxicity andantitumor activity of P38B-DM1 against CHO/dPDPN cells incomparison with P38B in vitro and in vivo, suggesting that
P38B-DM1 is applicable for antibody therapy against canine
cancers expressing dPDPN. Although the stability of our ADC
has not been checked in serum or in vivo, the toxicity afteradministration in vivo has not been observed. This suggests thata rapid release of drug (DM1) from ADC did not occur afteradministration. In principle, we assume the action of mechanism of our ADC, as follows. ADC will be incorporated into
the cells by endocytosis through the binding with PDPN. The
incorporated ADC will be degraded in late endosome generated with fusion with lysosome into drug (DM1)-attachedamino acid (Lys) or oligopeptides, which can work as a killing
agent through the inhibition of tubulin polymerization. Further
studies on antitumor activities against endogenous dPDPNexpressing tumors are necessary to obtain a more detailed
understanding of antibody therapy against canine cancers.
Acknowledgments

The authors thank Mikiko Yanagawa, Yoshimi Nakamura(Tohoku University); Akiko Harakawa (BIKAKEN), Chisaki
Imai, Nana Osako, Daichi Hamada (Kagoshima University)for excellent 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) underGrant Nos: JP19am0401013 (Y.K.), 19am0401002 (Y.I.),
JP19am0101078 (Y.K.), and JP19ae0101028 (Y.K.), and by
Japan Society for the Promotion of Science (JSPS) KAKENHI(Grant Nos: 17K07299, M.K.K; and 19K07705, Y.K.).
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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: January 13, 2020
Accepted: February 19, 2020