MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 5, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0019

Original Articles

Development of Core-Fucose-Deficient Humanized
and Chimeric Anti-Human Podoplanin Antibodies

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Mika K. Kaneko,1 Tomokazu Ohishi,2 Takuro Nakamura,1 Hiroyuki Inoue,2 Junko Takei,1Masato Sano,1 Teizo Asano,1 Yusuke Sayama,1 Hideki Hosono,1 Hiroyoshi Suzuki,3Manabu Kawada,2 and Yukinari Kato1,4

Podoplanin (PDPN), a 36-kDa type I transmembrane O-glycoprotein, is expressed in normal cells, including
renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, and in cancer
cells, including brain tumors and squamous cell lung carcinomas. PDPN activates platelet aggregation bybinding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and PDPN/CLEC-2 interaction facilitates blood/
lymphatic vessel separation. We previously produced an anti-human PDPN monoclonal antibody (mAb), clone
NZ-1 (rat IgG2a, lambda) and its rat-human chimeric mAbs (NZ-8/NZ-12), which neutralize PDPN/CLEC-2
interactions and inhibit platelet aggregation and cancer metastasis. In this study, we first developed a humanizedanti-human PDPN mAb, named as NZ-27. We further produced a core-fucose-deficient version of NZ-27,
named as P1027 and a core-fucose-deficient version of NZ-12, named as NZ-12f. We investigated the binding
affinity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), andantitumor activity of P1027 and NZ-12f. We demonstrated that the binding affinities of P1027 and NZ-12f
against LN319 (a human glioblastoma cell line) are 1.1 · 10-8 and 3.9 · 10-9 M, respectively. ADCC reporter
assays demonstrated that NZ-12f shows 1.5 times higher luminescence than P1027. Furthermore, NZ-12f
showed 2.2 times higher ADCC than P1027, whereas both P1027 and NZ-12f showed high CDC activities
against LN319 cells. Using LN319 xenograft models, P1027 and NZ-12f significantly reduced tumor development in an LN319 xenograft model compared with control human IgG. Treatment with P1027 and NZ-12f
may be a useful therapy for patients with PDPN-expressing cancers.
Keywords: PDPN, humanized antibody, P1027

Introduction

A

36-kDa type I transmembrane O-glycoprotein,podoplanin (PDPN)/Aggrus/T1a/gp36, is a platelet
aggregation-inducing factor. It is expressed in many cancers,
including malignant brain tumors, mesothelioma, and several squamous cell carcinomas.(1–10) Expression of PDPN
is associated with cancer metastasis, epithelial–mesenchymaltransition, malignant progression,(11–18) and clinical outcome.(19–21) Only a small and phenotypically distinct subset
of cells in solid tumors are considered tumor-initiating
cells (TICs) that are resistant to conventional therapies andresponsible for relapse.(22) Therefore, targeting TICs could
be useful for cancer therapy.(23) PDPN is reported to be a
TIC marker(24); thus, immunotherapy using specific antibodies against PDPN may eradicate TICs in cancers.

We previously produced a rat anti-human PDPN monoclonal antibody (mAb), NZ-1.(5) NZ-1 is highly specificand sensitive, and displays high binding affinity against
PDPN.(25,26) NZ-1 inhibited tumor cell-induced platelet aggregation and cancer metastasis.(12) NZ-1 is highly internalized into glioma cells, and is well accumulated into tumors
in vivo, making NZ-1 a suitable candidate for therapy against
malignant gliomas.(27) We constructed a single-chain antibody variable region fragment using NZ-1 (NZ-1-scFv).(28)NZ-1-scFv was then fused to Pseudomonas exotoxin A(NZ-1-scFv-PE38). NZ-1-scFv-PE38 demonstrated significant activity against glioblastoma and medulloblastoma
cells, and induced tumor growth delay in D2159MG and
D283MED using in vivo tumor models.
We previously produced rat-human chimeric anti-PDPN
antibodies (NZ-8 and NZ-12) from NZ-1.(29,30) Light chains

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Japan.
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
4
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
2
3

167

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168

of NZ-8 and NZ-12 are human kappa and human lambda,
respectively. NZ-12 possesses higher antibody-dependent
cellular cytotoxicity (ADCC) and complement-dependent
cytotoxicity (CDC) compared with NZ-8.(30) NZ-12, together
with human natural killer (NK) cells, showed antitumor
effects against malignant pleural mesothelioma orthotopic
xenograft mice.(31) Furthermore, radioimmunotherapy (RIT)with 90Y-labeled NZ-12 significantly suppressed tumor
growth in an NCI-H226 mesothelioma xenograft mousemodel, prolonging survival without loss of body weight orobvious adverse effects.(32)
Many studies have revealed that core-fucose-deficientantibodies are known to show higher ADCC activities.(33,34)
Similarly, we previously produced a core-fucose-deficientversion of anti-podocalyxin mAb (47-mG2a), which wasnamed as 47-mG2a-f, to augment its ADCC activities.(35) As
expected, 47-mG2a-f exhibited stronger ADCC activities than
47-mG2a against OSCC cell lines such as HSC-2 and SAS
cells, leading to higher antitumor activities in those xenograftmodels. Furthermore, we recently developed a core-fucosedeficient mouse-canine chimeric anti-dog PDPN mAb(P38B), named as P38Bf.(36) We compared ADCC between
P38B and P38Bf against Chinese hamster ovary (CHO)/
dPDPN cells. Although both P38B and P38Bf revealed
high ADCC activities against CHO/dPDPN cells, P38Bf
demonstrated significantly higher ADCC compared with
P38B, especially at low concentrations, indicating that corefucose-deficient mAbs are very useful for increasing ADCC
activities.
In this study, we produced a core-fucose-deficient version
of a humanized anti-human PDPN mAb (NZ-27), named as
P1027 and a core-fucose-deficient version of rat-humanchimeric anti-human PDPN mAb (NZ-12), named as NZ-12f,
and compared their binding affinity, ADCC/CDC activities,
and antitumor activity.
Materials and Methods

KANEKO ET AL.

frame sequences of VH in human Ig, and CH of human IgG1
were subcloned into the pCAG-Neo vector (FUJIFILM Wako
Pure Chemical Corporation). The CDR of VL in the NZ-1
antibody, frame sequences of VL in human Ig, and CL ofhuman lambda chain were subcloned into the pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation).
Antibody expression vectors of NZ-27 and NZ-12 weretransfected into BINDS-09 using the ExpiCHO-S Expression
System (Thermo Fisher Scientific, Inc.), respectively. Produced mAbs are named as P1027 and NZ-12f, respectively.
P1027 and NZ-12f were purified using Protein G Sepharose 4
Fast Flow (GE Healthcare UK, Ltd., Buckinghamshire,
England). Human IgG was purchased from Sigma-AldrichCorp. (St. Louis, MO).
Determination of the binding affinity by flow cytometry

LN319 cells (2 · 105 cells) were resuspended with 100 lLof serially diluted antibodies (P1027 and NZ-12f; 0.006–
100 lg/mL) followed by FITC-labeled anti-human IgG(Thermo Fisher Scientific, Inc.). Fluorescence data were collected using a cell analyzer (EC800; Sony Corp., Tokyo,
Japan). Dissociation constants (KD) were obtained by fitting
binding isotherms using built-in one-site binding models in
GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA).
ADCC reporter gene assay

We performed the FccRIIIa stimulation assay using an
ADCC Reporter Bioassay kit from Promega (Madison, WI),
following the manufacturer’s instructions.(37) LN319 was
inoculated into a 96-well white solid plate, 12,500 cells per
well. Anti-PDPN mAbs were serially diluted and added to
LN319 cells. Engineered, immortalized Jurkat T lymphocytes (Promega) expressing the FccRIIIa receptor (75,000
cells in 25 lL) were then added and cocultured with antibodytreated target cells at 37C for 6 h. Luminescence using the
Bio-Glo Luciferase Assay System (Promega) was measured
with a GloMax luminometer (Promega).

Cell lines

LN319 was purchased from Addexbio Technologies (San
Diego, CA). ExpiCHO-S was purchased from Thermo Fisher
Scientific, Inc., (Waltham, MA). BINDS-09 (FUT8-knocked
out ExpiCHO-S cells) was established in our previous
study.(36) LN319 was cultured in Dulbecco’s modified
Eagle’s medium (DMEM) medium (Nacalai Tesque, Inc.,
Kyoto, Japan), supplemented with 10% heat-inactivated fetal
bovine serum (Thermo Fisher Scientific, Inc.) at 37C in ahumidified atmosphere of 5% CO2 and 95% air. ExpiCHO-S
and BINDS-09 were cultured following manufacturer’s
instructions.
Antibodies

For the generation of a rat-human chimeric anti-human
PDPN mAb (NZ-12), appropriate VH of rat NZ-1 antibody
and CH of human IgG1 were subcloned into pCAG-Neo(FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan), and VL of rat NZ-1 antibody and CL of human lambda
chain were subcloned into pCAG-Ble vectors (FUJIFILM
Wako Pure Chemical Corporation). For creation of a humanized anti-human PDPN mAb (NZ-27), the complementarity determining region (CDR) of VH in the NZ-1 antibody,

Atibody-dependent cellular cytotoxicity

Human NK cells were purchased from Takara Bio, Inc.,(Shiga, Japan). Target cells were labeled with 10 lg/mL
Calcein AM (Thermo Fisher Scientific, Inc.) and resuspended
in the same medium. Target cells were plated in 96-wellplates (1 · 104 cells/well) and mixed with effector cells, antiPDPN antibodies, or control human IgG. After a 6.5-hour
incubation, Calcein AM release to the supernatant from eachwell was measured. Fluorescence intensity was determinedusing a microplate reader (Power Scan HT; BioTek Instruments, Winooski, VT) with excitation and emission wavelengths of 485 and 538 nm, respectively. Cytolytic activity(% of lysis) was calculated as % lysis = (E - S)/(M - S) · 100,
where E is the fluorescence of combined target and effector
cells, S is spontaneous fluorescence of target cells only, andM is maximum fluorescence measured after lysing all cellswith a buffer containing 0.5% Triton X-100, 10 mM Tris-HCl(pH 7.4), and 10 mM of EDTA.
Complement-dependent cytotoxicity

Target cells were labeled with 10 lg/mL Calcein AM andresuspended in the same medium. The target cells were plated

HUMANIZED ANTI-HUMAN PDPN

in 96-well plates (2 · 104 cells/well) and mixed with either
anti-PDPN antibodies or control human IgG and 10% of
rabbit complement. After a 6.5-hour incubation, the Calcein
AM release of supernatant from each well was measured.
Fluorescence intensity was determined using a microplate
reader (Power Scan HT) with an excitation wavelength of
485 nm and an emission wavelength of 538 nm. Cytolytic
activity (% of lysis) was calculated as % lysis = (E - S)/(M - S) · 100, where E is fluorescence of combined target andeffector cells, S is spontaneous fluorescence of target cells
only, and M is maximum fluorescence measured after lysing
all cells with a buffer containing 0.5% Triton X-100, 10 mM
Tris-HCl (pH 7.4), and 10 mM of EDTA.

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Antitumor activity of anti-PDPN antibodies
in xenografts of LN319

Sixteen 6-week-old female BALB/c nude mice were purchased from Charles River and used at 7 weeks of age. LN319
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). Mice were injected subcutaneously in the left flank (100 lL) with the suspension
mentioned earlier (5 · 106 cells). After day 1, 100 lg of
P1027, NZ-12f, and control human IgG in 100 lL PBS were
injected into mice through intraperitoneal injection (i.p.).
Additional antibodies were injected on days 8 and 16. Human169

NK cells (8.0 · 105 cells) were injected around the tumors
on days 1, 8, and 16. Twenty-one days after cell implantation,
all mice were euthanized by cervical dislocation and tumor
diameters and volumes were determined as previously
described.(38)
Statistical analyses

All data were expressed as mean – SEM. Statisticalanalysis used ANOVA and Tukey–Kramer’s test using R
statistical (R Foundation for Statistical Computing, Vienna,
Austria). p < 0.05 was adopted as a level of statisticalsignificance.
Results
Development of a core-fucose-deficient humanizedanti-PDPN mAb (P1027) and a core-fucose-deficient
rat-human chimeric mAb (NZ-12f)

In this study, we produced core-fucose-deficient humanized anti-PDPN (P1027) and rat-human chimeric antibodies(NZ-12f) (Fig. 1). Binding affinity of P1027 and NZ-12f
against LN319 cells was 1.1 · 10-8 and 3.9 · 10-9 M, respectively, indicating that both mAbs possess high affinity
for LN319 cells, but NZ-12f possesses higher binding activity(Fig. 2).

FIG. 1. Development of a core-fucose-deficient humanized anti-PDPN mAb (P1027) and a core-fucose-deficient rathuman chimeric mAb (NZ-12f). White box, derived from NZ-1 (rat IgG); gray box, derived from human IgG. mAb,
monoclonal antibody; PDPN, podoplanin.

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170

KANEKO ET AL.

FIG. 2. Determination of binding affinity using flow cytometry. LN319 (2 · 105 cells) was resuspended with 100 mL ofserially diluted P1027 (A) or NZ-12f (B) (0.006–100 mg/mL).

ADCC reporter gene assay

We used LN319 glioblastoma cells as target cells, andcompared luminescence between P1027 and NZ-12f. EC50s
for P1027 and NZ-12f were 149 and 14.3 ng/mL, respectively(Fig. 3A, B). At a concentration of 3.3 lg/mL, NZ-12f
showed 1.5 times higher luminescence than P1027 (Fig. 3C).
Thus, NZ-12f might display higher ADCC activity thanP1027.
ADCC and CDC mediated by anti-PDPN antibodies

To apply targeted therapy toward PDPN, we investigated
ADCC induction against PDPN-expressing cell lines by antiPDPN antibodies using human NK cells as effector cells.
NZ-12f exhibited ADCC activity (51% cytotoxicity, Fig. 4A)
and CDC activity (58% cytotoxicity, Fig. 4B) against LN319
cells; P1027 exhibited ADCC activity (23% cytotoxicity,
Fig. 4A) and CDC activity (53% cytotoxicity, Fig. 4B)
against LN319 cells, indicating that both NZ-12f and P1027
showed high CDC activities against LN319 cells (P1027 vs.
control, p < 0.01; NZ-12f vs. control, p < 0.01; P1027 vs.
NZ-12f, n.s.), whereas NZ-12f possesses 2.2 times higher

FIG. 3. ADCC bioreporter assay. (A, B) Dose dependency
of anti-PDPN mAbs in the ADCC reporter assay. P1027(A) and NZ-12f (B) were serially diluted and incubated with
the LN319 cells. As effector cells, engineered, immortalized
Jurkat T lymphocytes were cocultured. Luminescence usinga Bio-Glo Luciferase Assay System was measured with a
GloMax luminometer. (C) Comparison of luminescence between P1027 and NZ-12f in the ADCC bioreporter assay at aconcentration of 3.3 mg/mL. Asterisks indicate statistical significance (**p < 0.01, Tukey–Kramer’s test). ADCC, antibodydependent cellular cytotoxicity.
ADCC compared with P1027 (P1027 vs. control, n.s.; NZ-12f
vs. control, p < 0.01; P1027 vs. NZ-12f, p < 0.01).
Antitumor activity of P1027 and NZ-12f
in LN319 xenografts

Induction of cytotoxicity by P1027 and NZ-12f was investigated in vivo using LN319 cell xenografts in the flanks ofnude mice. P1027, NZ-12f, and control human IgG were
injected i.p. three times, on days 1, 8, and 16 after cell injection. Human NK cells were injected around tumors ondays 1, 8, and 16. Tumor formation was observed in miceDownloaded by Yukinari Kato from www.liebertpub.com at 10/21/20. For personal use only.

HUMANIZED ANTI-HUMAN PDPN

171

FIG. 5. Evaluation of antitumor activity of anti-PDPN antibodies (tumor volume). Tumor volume was measured inmice with LN319 xenografts. Asterisks indicate statisticalsignificance (*p < 0.05, **p < 0.01, n.s.: not significant, Tukey–
Kramer’s test).
the ability of antibodies to activate the ADCC reporter gene.
The ADCC reporter system consists of an Fc region of an
antibody that binds to the FcgRIIIa receptor expressed on anengineered immortalized Jurkat T-lymphocyte cell line.(37)This binding activates gene transcription through the nuclear
factor of the activated T cell pathway, inducing the expression of firefly luciferase. The FcgRIIIa stimulation assay canbe used to evaluate the ability of an antibody to activate the

FIG. 4. ADCC and CDC activities of anti-PDPN antibodies. (A) ADCC activities induced by human NK cells
against LN319 were determined. (B) CDC activities against
LN319 were determined. Asterisks indicate statistical significance (**p < 0.01, n.s.: not significant, Tukey–Kramer’s test).
CDC, complement-dependent cytotoxicity; NK, natural killer.
in every group. P1027 and NZ-12f treatment significantly
reduced tumor development compared with control mice ondays 3, 7, 10, 13, 16, and 21 (Fig. 5). No difference between
P1027 and NZ-12f was observed at any time. Weights oftumors from P1027 and NZ-12f-treated mice were significantly less than for tumors from IgG-treated control mice
(Fig. 6A). No difference in weights of tumors between P1027
and NZ-12f was found. Resected tumors are depicted in
Figure 6B. Total body weight was not significantly different
among the three groups (Fig. 7A). Mice on day 21 are depicted
in Figure 7B. Both P1027 and NZ-12f exert a significant antitumor effect against PDPN-expressing brain tumors.
Discussion

We previously produced a FUT8-knockout cell line
(BINDS-09) from ExpiCHO-S cells.(36) By using BINDS-09,
we easily produced core-fucose-deficient antibodies to enhance ADCC activity. In this study, we produced both a corefucose-deficient humanized anti-PDPN antibody (P1027) anda rat-human chimeric antibody (NZ-12f). Binding affinitiesof P1027 and NZ-12f against LN319 cells were 1.1 · 10-8
and 3.9 · 10-9 M (Fig. 2), of the affinity of NZ-12f against
LN319 is 2.8 times than the affinity of P1027. We next investigated whether this difference of binding affinity affects

FIG. 6. Evaluation of antitumor activity of anti-PDPN antibodies (tumor weight). (A) Tumor weight was measured fromexcised LN319 xenografts. (B) Resected tumors of LN319
xenografts on day 21. Values are mean – SEM. An asterisk
indicates statistical significance (*p < 0.05, n.s.: not significant,Tukey–Kramer’s test).

172

KANEKO ET AL.

(data not shown), suggesting that antitumor activities wereexerted mainly by CDC because both P1027 and NZ-12f
showed high CDC activities against LN319 cells (Fig. 4B).
We need to further perform in vivo experiments using theother xenograft models, such as lung cancers and malignant
mesotheliomas to confirm that humanized anti-PDPN mAbs
may be effective for patients with PDPN-expressing cancers.
Acknowledgment

We thank Ms. Akiko Harakawa for technical assistance inanimal experiments.
Author Disclosure Statement

No competing financial interests exist.

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Funding Information

This research was supported in part by AMED under Grant
Numbers: JP20am0401013 (Y.K.), JP20am0101078 (Y.K.),
JP20ae0101028 (Y.K.), and JP20bm1004001 (Y.K.), and by
JSPS KAKENHI Grant Numbers 20K16322 (M.S.),
17K07299 (M.K.K.), and 19K07705 (Y.K.).
References

FIG. 7. Evaluation of antitumor activity of anti-PDPN
antibodies (body weight). (A) Body weights of mice with
LN319 xenografts. n.s.: not significant. (B) Appearance oftreated mice on day 21.
ADCC reporter gene. NZ-12f displays 1.5 times higher activation of the ADCC reporter gene compared with P1027(Fig. 3), indicating that binding affinity is associated with theability of antibodies to activate the ADCC reporter gene. In
the ADCC and CDC assays, both P1027 and NZ-12f demonstrated high CDC activities, whereas NZ-12f showed 2.2
times higher ADCC activity compared with P1027 (Fig. 4),
which are compatible with results from binding affinity and
ADCC reporter assays.
In our previous studies, antitumor effects were observed
after treatment of malignant pleural mesothelioma orthotopic
xenograft mice with NZ-12 and human NK cells.(31) Furthermore, RIT with 90Y-labeled NZ-12 significantly suppressed tumor growth in an NCI-H226 xenograft mousemodel, and prolonged survival without obvious adverse effects.(32) Although P1027 showed lower binding affinity andlower ADCC activities to LN319 cells, antitumor activity ofP1027 for LN319 xenografts was equal to that of NZ-12f(Figs. 5 and 6). We further conducted additional in vivo experiments using three times injection of 10 mg antibodies toassess whether the antitumor activities were saturated whenthree times injection of 100 mg was employed. However, weobserved no difference of antitumor activities between P1027
and NZ-12f using three times injection of 10 mg antibodies

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KANEKO ET AL.

38. Kato Y, Kunita A, Abe S, Ogasawara S, Fujii Y, Oki H,
Fukayama M, Nishioka Y, and Kaneko MK: The chimericantibody chLpMab-7 targeting human podoplanin suppresses
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: April 17, 2020
Accepted: August 20, 2020