Cancer Medicine

Open Access

ORIGINAL RESEARCH

Antitumor activity of chLpMab-­2, a human–mouse chimericcancer-­specific antihuman podoplanin antibody, via
antibody-­dependent cellular cytotoxicity
Mika K. Kaneko1, Shinji Yamada1, Takuro Nakamura1, Shinji Abe2,3, Yasuhiko Nishioka3, Akiko Kunita4,
Masashi Fukayama4, Yuki Fujii1, Satoshi Ogasawara1,5 & Yukinari Kato1,6,7
1Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
2Department of Clinical Pharmacy Practice Pedagogy, Graduate School of Biomedical Sciences, Tokushima University, 1-78-1 Sho-machi, Tokushima

770-8505, Japan
3Department of Respiratory Medicine and Rheumatology, Graduate School of Biomedical Sciences, Tokushima University, 3-18-15 Kuramoto-cho,
Tokushima 770-8503, Japan
4Department of Pathology, Graduate School of Medicine, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
5Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan
6Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi
980-8575, Japan
7Project of Antibody Drug Development, New Industry Creation Hatchery Center, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan

Keywords
Antibody-dependent cellular cytotoxicity,chimeric antibody, human podoplanin,
monoclonal antibody
Correspondence
Yukinari Kato, Tohoku University Graduate
School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai, Miyagi 980-8575, Japan.
Tel/Fax: +81-22-717-8207;
E-mail: yukinari-k@bea.hi-ho.ne.jp
Funding Information
This work was supported in part by the Basic
Science and Platform Technology Program for
Innovative Biological Medicine from Japan
Agency for Medical Research andDevelopment, AMED (Y.K.), by theTranslational Research Network Program from
AMED (Y.K.), by the Platform for Drug
Discovery, Informatics, and Structural Life
Science (PDIS) from AMED (Y.K.), by Projectfor utilizing glycans in the development ofinnovative drug discovery technologies from
AMED (Y.K.), by the Regional Innovation
Strategy Support Program from the Ministryof Education, Culture, Sports, Science andTechnology (MEXT) of Japan (Y.K.), by JSPS
KAKENHI Grant Number 26440019 (M.K.K.),
16K08372 (S.A.), and 16K10748 (Y.K.).

Abstract
Human podoplanin (hPDPN), a platelet aggregation-­inducing transmembrane
glycoprotein, is expressed in different types of tumors, and it binds to C-­type
lectin-­like receptor 2 (CLEC-­2). The overexpression of hPDPN is involved ininvasion and metastasis. Anti-­hPDPN monoclonal antibodies (mAbs) such as
NZ-­
1 have shown antitumor and antimetastatic activities by binding to theplatelet aggregation-­stimulating (PLAG) domain of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-­2, using the cancer-­specificmAb (CasMab) technology. In this study we developed chLpMab-­2, a human–
mouse chimeric anti-­hPDPN antibody, derived from LpMab-­2. chLpMab-­2 wasproduced using fucosyltransferase 8-­
knockout (KO) Chinese hamster ovary(CHO)-­
S cell lines. By flow cytometry, chLpMab-­
2 reacted with hPDPN-­
expressing cancer cell lines including glioblastomas, mesotheliomas, and lungcancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab-­
2 exhibitedhigh antibody-­dependent cellular cytotoxicity (ADCC) against PDPN-­expressing
cells, despite its low complement-­dependent cytotoxicity. Furthermore, treatment
with chLpMab-­2 abolished tumor growth in xenograft models of CHO/hPDPN,
indicating that chLpMab-­2 suppressed tumor development via ADCC. In conclusion, chLpMab-­2 could be useful as a novel antibody-­based therapy against
hPDPN-­expressing tumors.

Received: 3 January 2017; Revised: 28
January 2017; Accepted: 7 February 2017
doi: 10.1002/cam4.1049© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.

1

Chimeric Anti-­PDPN Antibody ChLpMab-­2

Introduction
Human podoplanin (hPDPN), also known as hT1α, hAggrus, or gp36, is expressed in many types of cancers,
such as malignant brain tumors, malignant mesotheliomas,lung cancers, esophageal cancers, testicular tumors, bladdercancers, osteosarcomas, and fibrosarcomas [1–15]. hPDPN
expression is observed only in squamous cell carcinomasof lung and esophageal cancers, indicating that hPDPN
exhibits histological type-­specific expression. The expression of hPDPN in cancer-­associated fibroblasts contributes
to a poor prognosis [16–21]. C-­type lectin-­like receptor
2 (CLEC-­
2) is an endogenous receptor of hPDPN [22,23]. CLEC-­
2 binds to hPDPN through residues Glu47
and Asp48 within its platelet aggregation-­stimulating(PLAG) domain and to α-­2,6-­linked sialic acid, which is
attached to Thr52 [24].
Membrane proteins could be targeted by antibody-­based
therapy if (1) they possess cancer-­specific mutations outside
the membrane [25], (2) they are overexpressed in cancers
rather than in normal tissues [26–28], or (3) they are
posttranslationally modified by phosphorylation or glycosylation [29, 30]. Although many anti-­hPDPN monoclonal
antibodies (mAbs) are commercially available, most ofthem react with the N-­terminus of hPDPN, and they do
not fulfill the above-­mentioned criteria [6, 31–35]. hPDPN
is highly expressed in normal lymphatic endothelial cells(LECs) and normal lung type-­I alveolar cells at the samelevel as in cancer cells. We previously produced a rat
anti-­
hPDPN mAb (NZ-­
1), which detects hPDPN withhigh specificity and sensitivity [6, 10, 31]. NZ-­1 is efficiently internalized by glioma cell lines and accumulates
in tumors in vivo; therefore, it has been suggested to bea suitable candidate for therapy against malignant gliomas[5, 10]. Moreover, NZ-­1 inhibits tumor cell-­induced platelet
aggregation and tumor metastasis [23]. NZ-­
1 mediates
antibody-­
dependent cellular cytotoxicity (ADCC) andcomplement-­dependent cytotoxicity (CDC) against tumor
cells that express hPDPN [36]. Furthermore, human–rat
chimeric antibodies, such as NZ-­
8 and NZ-­
12, exhibit
high ADCC and CDC in vitro, and they show very highantitumor activities and neutralizing capabilities [36, 37].
Nevertheless, these chimeric mAbs are not cancer-­specific;
therefore, cancer-­specific anti-­hPDPN chimeric or humanized antibodies should be developed to prevent unfavorable
side effects.
We have developed the original technology to produce
cancer-­specific mAbs (CasMabs) against hPDPN [29,38–46]. The established LpMab-­2 recognized the aberrantO-­glycosylation and Thr55–Leu64 peptide of hPDPN [29].
LpMab-­2 reacted with hPDPN-­expressing cancer cells but
not with normal cells, as shown by flow cytometry andimmunohistochemistry. Therefore, LpMab-­
2 is an2

M. K. Kaneko et al.

anti-­
hPDPN CasMab that can be used for molecular-­targeted therapy against hPDPN-­
expressing cancers. In
immunohistochemistry, less than 10% of hPDPN-­
expressing cancer cells were recognized by LpMab-­2. In
contrast, another anti-­hPDPN mAb LpMab-­7 reacted withboth hPDPN-­expressing cancer cells and normal cells [42].
We identified the minimum epitope of LpMab-­7 as Arg79–
Leu83 of hPDPN using ELISA, Western-­blot, and flow
cytometry. We further produced a human–mouse chimericanti-­hPDPN mAb, chLpMab-­7 [40]. chLpMab-7 showed
ADCC and CDC, and inhibited the growth of hPDPN-­
expressing tumors in vivo.
In this study, we developed and characterized chLpMab­2, a human–mouse chimeric anti-­hPDPN antibody derived
from LpMab-­2.

Materials and Methods
Antibodies
LpMab-­
2, a mouse anti-­
hPDPN mAb (IgG1, kappa), wasdeveloped as previously described [29]. Human IgG waspurchased from Beckman Coulter, Inc. (Fullerton, CA). To
generate human–mouse chimeric anti-­hPDPN (chLpMab-­2),
appropriate VH and VL cDNAs of mouse LpMab-­2 and CH
and CL of human IgG1 were subcloned into pCAG-­Ble andpCAG-­Neo vectors (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan), respectively. CHO-­
S/fucosyltransferase 8(FUT8)-­KO (PDIS-­5) cell lines were generated by transfecting CRISPR/Cas9 plasmids that targeted FUT8 (Target ID:
HS0000547010; Sigma-­Aldrich Corp., St. Louis, MO) into
CHO-­S cells (Thermo Fisher Scientific Inc., Waltham, MA)
using a Gene Pulser Xcell electroporation system. PDIS-­
5
cells were screened using Aleuria aurantia lectin. Antibody
expression vectors were transfected into PDIS-­5 using the
Lipofectamine LTX reagent (Thermo Fisher Scientific Inc.).
Stable transfectants of PDIS-­5/chLpMab-­2 were selected bycultivating the transfectants in a medium containing 0.5 mg/
mL of both geneticin and zeocin (InvivoGen, San Diego,
CA). PDIS-­
5/chLpMab-­
2 cells were cultivated in CHO-­S-­
SFM II medium (Thermo Fisher Scientific Inc.). chLpMab-2
was purified using Protein G-­Sepharose (GE healthcare Bio-­
Sciences, Pittsburgh, PA).

Cell lines
The cell lines LN229, HEK-­
293T, NCI-­
H226, Met-­
5A,
Chinese hamster ovary (CHO)-­K1, and P3U1 were obtained
from the American Type Culture Collection (Manassas,
VA). The LN319 cell line was provided by Prof. KazuhikoMishima (Saitama Medical University, Saitama, Japan) [47].
Human LECs and PC- 10 cells were purchased from
Cambrex Corp. (Walkersville, MD) and Immuno- Biological© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Chimeric Anti-­PDPN Antibody ChLpMab-­2

M. K. Kaneko et al.

Laboratories Co., Ltd. (Gunma, Japan), respectively. LN229
and CHO-­K1 cells were transfected with hPDPN plasmids
using Lipofectamine 2000 (Thermo Fisher Scientific Inc.)
according to the manufacturer’s instructions [29]. The
LN319/hPDPN-­
KO cell line (PDIS-­
6) was generated bytransfection using CRISPR/Cas9 plasmids (Target ID:
HS0000333287) that targeted PDPN (Sigma-­
Aldrich, St.
Louis, MO), as previously described [48]. The cell lines
CHO-­S/GnT-­1-­KO (PDIS-­9) and CHO-­S/SLC35A1-­KO(PDIS-­
14) were generated by transfecting TALEN and
CRISPR/Cas9 plasmids, respectively. The former plasmid-­targeted hsMgat1 (Wako Pure Chemical Industries Ltd.)and the latter targeted SLC35A1 (Target ID: HS0000168432;
Sigma-­Aldrich) [49].
The CHO-­K1, CHO/hPDPN, NCI-­H226, PC-­10, and P3U1
cells were cultured in RPMI 1640 medium containingL-­glutamine (Nacalai Tesque, Inc., Kyoto, Japan). The LN229,
LN229/hPDPN, LN319, HEK-­293T, and PDIS-­6 cells were
cultured at 37°C in a humidified atmosphere containing
5% CO2 in Dulbecco’s Modified Eagle’s Medium containingL-­glutamine (Nacalai Tesque, Inc.) and 10% heat-­inactivatedfetal bovine serum (FBS) (Thermo Fisher Scientific Inc.).
CHO-­
S, PDIS-­9, and PDIS-­
14 were cultured in CHO-­S-­
SFMII medium (Thermo Fisher Scientific Inc.). LECs were
cultured in the endothelial cell medium EGM-­
2MV supplemented with 5% FBS (Cambrex Corp.). All media contained 100 U/mL of penicillin, 100 μg/mL of streptomycin,
and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.).

RPMI 1640 supplemented with 10% FBS, 51Cr-­labeled target
cells were seeded in 96-­well plates in triplicate. Humanperipheral blood MNCs and chLpMab-­2 or control human
IgG were added to the cells. After 6 h of incubation, 51Cr
released from cells into the supernatant (100 μL) was measured using a gamma counter (PerkinElmer, Waltham, MA).
The percentage of cytotoxicity was calculated using thefollowing formula: % specific lysis = (E − S)/(M − S) ×100, where E is the release in the test sample, S is the
spontaneous release, and M is the maximum release.

CDC
CDC was evaluated by the 51Cr release assay as previously
described [9]. The CHO/hPDPN cells (target cells) wereincubated with 51Cr-­sodium chromate (0.1 μCi) for 1 h
at 37°C. After incubation, the cells were washed with RPMI
1640 supplemented with 10% FBS. The 51Cr-­labeled cells
were incubated with baby rabbit complement (Cedarlane,
Ontario, Canada) at a dilution of 1:32 and chLpMab-­
2(0.01–10 μg/mL) or control human IgG (0.01–10 μg/mL)
for 3 h in 96-­well plates. After incubation, 51Cr in the
supernatant was measured using a gamma counter. The
percentage of cytotoxicity was calculated using the following formula: % specific lysis = (E − S)/(M − S) × 100,
where E is the release in the test sample, S is the spontaneous release, and M is the maximum release.

Antitumor activity of anti-­hPDPN antibodies
Flow cytometry
The cell lines were harvested after brief exposure to 0.25%
trypsin/1 mmol/L EDTA (Nacalai Tesque, Inc.). Afterwashing with 0.1% BSA in PBS, the cells were treatedwith primary mAbs for 30 min at 4°C, followed by treatment with FITC-labeled goat anti-human IgG (Thermo
Fisher Scientific Inc.). Fluorescence data were acquired
using Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

Preparation of effector cells
Effector cells were prepared as previously described [9].
Human peripheral blood mononuclear cells (MNCs) wereobtained from leukocytes, which were separated from theperipheral blood of healthy donors. The study with humansubjects was approved by the Ethics Committee ofTokushima University.

ADCC
ADCC was determined with the 51Cr release assay [9].
Target cells were incubated with 0.1 μCi of 51Cr-­sodium
chromate at 37°C for 1 h. After washing three times with© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

CHO/hPDPN cells were trypsinized and washed with PBS.
The cell density was adjusted with PBS to 5.0 × 107 cells/
mL, and 100 μL/animal of the cell suspension was subcutaneously inoculated into BALB/c nude mice. After
1 day, 100 μL of 1 mg/mL of chLpMab-­
2 and human
IgG were injected into the peritoneal cavity of mice, oncea week for 4 weeks (control group, n = 6; chLpMab-­2group, n = 6). Human NK cells (5.0 × 105 cells, Takara
Bio Inc., Shiga, Japan) were injected around the tumors
4 and 11 days after cell inoculation. The tumor diameter
was measured every 3–4 days and was calculated using
the following formula: volume = W2 × L/2, where W is
the short diameter and L is the long diameter. The mice
were euthanized 21 days after cell implantation.

Statistical analysis
All data were expressed as means ± SEMs. Student’s t­-­test,
Mann–Whitney U-­
test, one-­way ANOVA followed by
Tukey–Kramer multiple comparisons, and two-­way
ANOVA were performed as appropriate. P values less than
0.05 were considered to be statistically significant. All
statistical tests were two-­sided.
3

Chimeric Anti-­PDPN Antibody ChLpMab-­2

Results
Production of chLpMab-­2
We developed chLpMab-­2 from a mouse mAb, LpMab-­2.
chLpMab-­2 reacted with LN229/hPDPN cells as revealedby flow cytometry (Fig. 1A). chLpMab-­
2 detected

Figure 1. Flow cytometric analysis using chLpMab-2 to detect
hPDPN expression. (A) LN229 and LN229/hPDPN cells were treatedwith chLpMab-­2 (1 μg/mL, red), chLpMab-­7 (1 μg/mL, blue), and PBS(black) for 30 min at 4°C, followed by treatment with antihuman IgG-­
FITC. (B) LN319 and LN319/hPDPN-­KO cells (PDIS-­6) were treated with
chLpMab-­2 (1 μg/mL, red), chLpMab-­7 (1 μg/mL, blue), and PBS (black)for 30 min at 4°C, followed by treatment with antihuman IgG-­FITC.
Fluorescence data were collected using Cell Analyzer EC800.

4

M. K. Kaneko et al.

endogenous hPDPN in glioblastoma cell line LN319 andnot in the LN319/hPDPN-­
KO cells (PDIS-­
6) (Fig. 1B).
Our results showed that chLpMab-­2 was specific against
hPDPN.

Figure 2. Flow cytometric analysis using chLpMab-2 to detect
PDPN expression in human cancer and normal cells. (A) Humancancer cell lines such as lung squamous cell carcinoma (PC-­10) andmesothelioma (NCI-­H226) were treated with chLpMab-­2 (1 μg/mL, red),chLpMab-­7 (1 μg/mL, blue), and PBS (black) for 30 min at 4°C, followed
by treatment with antihuman IgG-­FITC. (B) Normal human cell lines
such as mesothelial cells (Met5A) and renal epithelial cells (HEK-­293T)
were treated with chLpMab-­2 (1 μg/mL, red), chLpMab-­7 (1 μg/mL,blue), and PBS (black) for 30 min at 4°C, followed by treatment withantihuman IgG-­FITC. Fluorescence data were acquired using Cell
Analyzer EC800.

© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Chimeric Anti-­PDPN Antibody ChLpMab-­2

M. K. Kaneko et al.

Flow cytometric analyses of chLpMab-­2 incancer and normal cell lines
hPDPN is expressed in cancers such as brain tumors andmesotheliomas. By flow cytometry, chLpMab-­
2 detectedendogenous PDPN in human cancer cell lines such asPC-­10 of lung squamous cell carcinoma and NCI-­H226
of mesothelioma (Fig. 2A). A positive control, chLpMab-­7,
detected PDPN expression in two cancer cell lines (Fig. 2A).
Because of low hPDPN expression in NCI-­
H226 [29],the reaction of chLpMab-­2 against NCI-­H226 was much
lower than that of chLpMab-­7. In contrast, chLpMab-­2
failed to detect the expression of hPDPN in normal cells
such as renal epithelial cells (HEK-­293T) and the mesothelial cell line Met-­5A (Fig. 2B). However, chLpMab-­7
reacted with these cells (Fig. 2B), indicating that chLpMab-­2
was cancer-­specific. These results are consistent with thoseof our previous study [29].

Characterization of chLpMab-­2 using glycan-­deficient cell lines
Previous studies have shown that hPDPN is O-­glycosylated
and not N-­glycosylated [6, 24, 50–52]. We used a GnT-­
1-­KO cell line (CHO-­S/GnT-­1-­KO, PDIS-­9) and a CMP-­
sialic acid transporter (SLC35A1)-­KO cell line (CHO-­S/
SLC35A1-­KO, PDIS-­14) [49] to characterize chLpMab-­2.

As shown in Figure 3B, chLpMab-­7 reacted with CHO-­S/
hPDPN, PDIS-­9/hPDPN, and PDIS-­14/hPDPN cells transfected with the hPDPN expression vector. In contrast,
chLpMab-­2 reacted with the CHO-­S/hPDPN and PDIS-­9/hPDPN cells and not with the PDIS-­
14/hPDPN cells(Fig. 3A). This result agrees with that of our previous
study [29] that the epitope of chLpMab-­2 includes sialic
acid.

ADCC and CDC
LpMab-­
2 was previously determined to belong to themouse IgG1 subclass that failed to induce ADCC and
CDC [29]. Therefore, in this study, we converted LpMab-­2
to chLpMab-­2 of human IgG1 to study ADCC and CDC[40]. As shown in Figure 4A, chLpMab-­2 showed ADCC
against the LN319, PC-­10, and NCI-­H226 cell lines. The
CHO/hPDPN and CHO-­K1 cells were used to study the
specificity of chLpMab-­
2 against hPDPN. As shown in
Figure 4B, chLpMab-­2 showed ADCC against the CHO/
hPDPN cells and not against the hPDPN-­negative CHO-­
K1 cells. Furthermore, chLpMab-­2-­induced ADCC against
CHO/hPDPN in a dose-­dependent manner (Fig. 5A). In
contrast, chLpMab-­
2 shows weak CDC against CHO/
hPDPN cells (Fig. 5B) and against LN319, PC-­10, and
NCI-­H226 cell lines (data not shown).

Figure 3. Flow cytometric analysis using chLpMab-­2 to detect hPDPN expression in N-­glycan and sialic acid-­deficient cells. CHO-­S, CHO-­S/hPDPN,
PDIS-­9/hPDPN, and PDIS-­14/hPDPN cells were reacted with chLpMab-­2 (A, 1 μg/mL; red), chLpMab-­7 (B, 1 μg/mL; blue), and PBS (black) for 30 min
at 4°C, followed by treatment with antihuman IgG-­FITC. Fluorescence data were acquired using Cell Analyzer EC800.

© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

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Chimeric Anti-­PDPN Antibody ChLpMab-­2

Antitumor activity of chLpMab-­2
To study the antitumor activity of chLpMab-­2 on primarytumor growth in vivo, the CHO/hPDPN cells were subcutaneously implanted into the flanks of nude mice. The
LN319, PC-­10, and NCI-­H226 cells were not tested because
they were not suitable for xenograft models. chLpMab-­2
and control human IgG were injected into the peritoneal
cavity of mice, once weekly for 4 weeks (n = 6 each),and human NK cells were injected twice around the tumors.
Tumor formation was observed in mice from the control
and treated groups. However, chLpMab-­
2 significantly
reduced tumor development compared with control human
IgG (Fig. 6A and B). The tumor volume was significantly
reduced by chLpMab-­2 treatment on day 15, 18, and 21(Fig. 6C). These results indicate that the administrationof chLpMab-­
2 with the NK cells inhibited the primarytumor growth of the CHO/hPDPN cells in vivo.

M. K. Kaneko et al.

recognized not only the Thr55–Leu64 peptide of hPDPN
but also an aberrant O-­
glycosylated hPDPN, which is
attached to Thr55 or Ser56 of hPDPN [48]. LpMab-­2
reacted with hPDPN-­expressing cancer cells and not withnormal cells, as revealed by flow cytometry and immunohistochemistry [29]; therefore, LpMab-­
2 is an anti-­
hPDPN CasMab that is potentially advantageous for
antibody-­based molecular-­targeted therapy against hPDPN-­
expressing cancers. However, LpMab-­2 is a mouse IgG1;
therefore, it cannot be used to study ADCC and CDC
against hPDPN-­expressing cancers.
To our knowledge, herein we developed the first cancer-­
specific human–mouse chimeric anti-­
hPDPN antibody(chLpMab-­2) from LpMab-­2. Previously, we had developed
a human–mouse chimeric anti-­
hPDPN antibody

Discussion
We previously produced LpMab-­2 (mouse IgG1, kappa),
one of the CasMabs against hPDPN [29]. LpMab-­
2

Figure 4. ADCC of chLpMab-­2 against hPDPN-­expressing cell lines. (A)
ADCC of chLpMab-­2 using human MNCs against (A) LN319, PC-­10,and NCI-­H226 and (B) CHO and CHO/hPDPN were evaluated by a 6-­h51Cr release assay in the presence of 1 μg/mL of antibody with an E/Tratio of 100. **P < 0.01

6

Figure 5. ADCC and complement-­dependent cytotoxicity (CDC) of
chLpMab-­2 against CHO/hPDPN cells. (A) ADCC of chLpMab-­2 using
human MNCs against CHO/hPDPN was evaluated by a 6-­h 51Cr releaseassay in the presence of 0.01–10 μg/mL of antibody with an E/T ratio of100. (B) CDC of chLpMab-­2 using baby rabbit complement against
CHO/hPDPN was evaluated by 51Cr release assay in the presence of
0.01–10 μg/mL of antibody. *P < 0.05; **P < 0.01

© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

M. K. Kaneko et al.

Figure 6. Antitumor effects of chLpMab-­2 on CHO/hPDPN tumor
development. CHO/hPDPN cells (3 × 106 cells/100 μL) were
subcutaneously inoculated into BALB/c nude mice. After 1 day, 100 μgof chLpMab-­2 or control human IgG was injected into the peritoneal
cavity of the mice. The antibodies were injected once weekly for
4 weeks (control group: n = 6; chLpMab-­2 group: n = 6). The tumor
diameter was measured every 3-­4 days and was calculated using thefollowing formula: tumor volume = W2 × L/2, where W is the short
diameter and L is the long diameter. (A) Comparison of the tumor size
and tumor incidence in nude mice (day 21). (B) Comparison of thetumor size (day 21). (C) Primary tumor growth in human IgG and
chLpMab-2-treated mice. **P < 0.01; ****P < 0.001 with two-­way
ANOVA.

(chLpMab-­7) [40] and human–rat chimeric anti-­hPDPN
antibodies such as NZ-­
8 [36] and NZ-­
12 [37]. Other
groups have reported the development of human–mousechimeric anti-­
hPDPN/hAggrus antibodies that were notcancer-­specific [32, 33]. Because hPDPN is expressed inmany normal organs such as the lung and kidney,© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Chimeric Anti-­PDPN Antibody ChLpMab-­2non-­CasMabs against hPDPN are not suitable for antibody-­
based molecular targeting therapy.
In this study, we used FUT8-­KO CHO-­S cells (PDIS-­5)
to express afucosylated chLpMab-­2. Afucosylated antibodies
are known to exhibit high ADCC [53]. Consistent withthe literature, the ADCC of afucosylated chLpMab-­2 was
3.4 times higher than that of fucosylated chLpMab-­2 (datanot shown). In contrast, afucosylated chLpMab-­2 showeda lower CDC (Fig. 5B) than fucosylated chLpMab-­2 (datanot shown). Gasdaska et al. have reported that the higher
ADCC of afucosylated rituximab suggests an improvementin effectiveness and potency, however, its lower CDC may
mitigate infusion toxicity [54]. They concluded that afucosylated rituximab was clinically better than fucosylated
rituximab. In contrast, Niwa et al. have reported that
fucose depletion can provide a panel of IgGs (IgG1, IgG2,
IgG3, and IgG4) with enhanced ADCC but that none of
the IgGs affected CDC [53].
In our previous study, a human–mouse chimeric anti-­
hPDPN mAb showed CDC for antitumor activity in theabsence of human NK cells in a mouse xenograft model[36]. In this study, chLpMab-­2 exhibited ADCC for antitumor activity in the xenograft model with added NK
cells [9]. Our results indicate that ADCC is important
to induce the antitumor activity of anti-­hPDPN CasMabin the xenograft model for two reasons: i) fucosylated
chLpMab-­2, which was expressed in CHO-­K1 cells, showed
high CDC but failed to induce enough ADCC and antitumor activity against hPDPN-­
expressing tumors (datanot shown) and ii) afucosylated chLpMab-­2 did not show
enough CDC in vitro (Fig. 5B) but exhibited higher ADCC
in vitro (Fig. 5A) and antitumor activity against hPDPN-­
expressing tumors (Fig. 6).
Taken together, chLpMab-­2 may be useful as a novel
antibody-­based therapy against hPDPN-­expressing tumors
with no unexpected side effects.

Acknowledgments
We thank Miyuki Yanaka, Noriko Saidoh, and Kanae
Yoshida. for their excellent technical assistance. We also
thank Takeshi Murata, Hiroaki Uchida, and Hideaki Tahara
for their specialized advice. This work was supported inpart by the Basic Science and Platform Technology Programfor Innovative Biological Medicine from Japan Agency
for Medical Research and Development, AMED (Y.K.),
by the Translational Research Network Program from
AMED (Y.K.), by the Platform for Drug Discovery,
Informatics, and Structural Life Science (PDIS) from AMED
(Y.K.), by Project for utilizing glycans in the developmentof innovative drug discovery technologies from AMED
(Y.K.), by the Regional Innovation Strategy Support
Program from the Ministry of Education, Culture, Sports,
7

Chimeric Anti-­PDPN Antibody ChLpMab-­2

Science and Technology (MEXT) of Japan (Y.K.), by JSPS
KAKENHI Grant Number 26440019 (M.K.K.), 16K08372(S.A.), and 16K10748 (Y.K.). This work was performedin part under the Cooperative Research Program of Institutefor Protein Research, Osaka University, CR-­16-­05 and by
the Grant for Joint Research Project of the Institute ofMedical Science, the University of Tokyo. The authors
would like to thank Enago (www.enago.jp) for the English
language review.

Conflict of Interest
None declared.
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© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.