MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 1, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2016.0047

Chimeric Anti-Human Podoplanin Antibody NZ-12
of Lambda Light Chain Exerts Higher Antibody-Dependent
Cellular Cytotoxicity and Complement-Dependent
Cytotoxicity Compared with NZ-8 of Kappa Light Chain
Mika K. Kaneko,1 Shinji Abe,2,3 Satoshi Ogasawara,1,4,5 Yuki Fujii,1 Shinji Yamada,1 Takeshi Murata,4,5
Hiroaki Uchida,6 Hideaki Tahara,6 Yasuhiko Nishioka,3 and Yukinari Kato1

Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal
epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells,
including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding toC-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/
lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody(mAb), clone NZ-1 (rat IgG2a, lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet
aggregation and cancer metastasis. Human–rat chimeric antibody, NZ-8, was previously developed using variable
regions of NZ-1 and human constant regions of heavy chain (IgG1) and light chain (kappa chain). Although NZ-8
showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)
against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein,
we produced a novel human–rat chimeric antibody, NZ-12, the constant regions of which consist of IgG1 heavy
chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was muchhigher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against
glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12
could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.
Keywords: human podoplanin, chimeric antibody, monoclonal antibody, lambda chain, kappa chain

for cancer therapy.(23) Podoplanin has been reported to be a
TIC marker(24); immunotherapy using specific antibodies
reactive to podoplanin may eradicate TICs in cancers.
We previously produced a rat anti-human podoplanin
monoclonal antibody (mAb), NZ-1.(5) NZ-1 possesses highspecificity, high sensitivity, and high binding-affinity against
podoplanin.(25,26) Because NZ-1 is highly internalized into
glioma cell lines, and is also well accumulated into tumors
in vivo, NZ-1 is a suitable candidate for therapy against
malignant gliomas.(27) By its neutralizing activity, NZ-1 also
inhibited the tumor cell-induced platelet aggregation andcancer metastasis.(12) We constructed a single-chain antibody
variable region fragment using NZ-1 (NZ-1-scFv).(28) NZ-1scFv was then fused to Pseudomonas exotoxin A (NZ-1-

Introduction

A

type I transmembrane 36-kDa glycoprotein, podoplanin (PDPN/Aggrus/T1a/gp36), is a platelet aggregationinducing factor, and its expression has been reported in many
cancers such as malignant brain tumors, mesotheliomas, andseveral squamous cell carcinomas.(1–10) Expression of podoplanin has been reported to be associated with cancer
metastasis, epithelial-mesenchymal transition, malignant
progression,(11–18) and clinical outcome.(19–21) Only a small
and phenotypically distinct subset of cells in solid tumors is
considered as tumor-initiating cells (TICs),(22) which areresistant to conventional therapies and responsible for relapse. Therefore, targeting TICs could be a useful approach1

Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Clinical Pharmacy Practice Pedagogy, Graduate School of Biomedical Sciences, Tokushima University, Tokushima, Japan.
3
Department of Respiratory Medicine and Rheumatology, Graduate School of Biomedical Sciences, Tokushima University, Tokushima, Japan.
4
Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan.
5
Molecular Chirality Research Center, Chiba University, Chiba, Japan.
6
Division of Bioengineering, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
2

25

26

scFv-PE38). NZ-1-scFv-PE38 exhibited significant activity
against glioblastoma and medulloblastoma cells, and demonstrated tumor growth delay in D2159MG and D283MED
in vivo tumor models.
Furthermore, we produced a human–rat chimeric antipodoplanin antibody (NZ-8) from NZ-1.(29) NZ-8 possesses
high antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC). Furthermore,
NZ-8 inhibits the growth of podoplanin-expressing tumors
in vivo, and also suppresses hematogenous metastasis of
podoplanin-expressing tumors. In contrast, the binding affinity of NZ-8 is lower than that of NZ-1.
Herein, we produced a novel human–rat chimeric antibody, NZ-12, the constant regions of which consist of IgG1
heavy chain and lambda light chain.
Materials and Methods
Cell lines

CHO-K1 was obtained from the American Type Culture
Collection (ATCC, Manassas, VA). LN319 and D397 were
provided by Dr. Kazuhiko Mishima (Saitama Medical University, Saitama, Japan) and Dr. Darell D. Bigner (Duke
University Medical Center, Durham, NC), respectively. PC10 cells were purchased from Immuno-Biological Laboratories Co., Ltd. (Gunma, Japan). CHO-K1 and PC-10 were
cultured in RPMI 1640 medium (Nacalai Tesque, Inc.,
Kyoto, Japan), and LN319 and D397 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated fetal
bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37C
in a humidified atmosphere of 5% CO2 and 95% air.
Antibodies

A rat anti-human podoplanin mAb (NZ-1) and a human–rat chimeric anti-human podoplanin antibody (NZ-8) weredeveloped as described previously.(5,27,29,30) For the generation of human–rat chimera anti-human podoplanin (NZ-12),
the appropriate VH of a rat NZ-1 antibody and CH of human
IgG1 were subcloned into the pCAG-Neo (Wako Pure Chemical Industries Ltd., Osaka, Japan), and VL of a rat NZ-1
antibody and CL of human lambda chain were subcloned into
pCAG-Ble vectors (Wako Pure Chemical Industries Ltd.).
Antibody expression vectors were transfected into CHO-K1
cells using Lipofectamin LTX (Thermo Fisher Scientific,Inc.). Stable transfectants of CHO/NZ-12 were selected bycultivating the transfectants in medium containing 1 mg/mL
G418 (Wako Pure Chemical Industries Ltd.) and 0.5 mg/mL
Zeocin (InvivoGen, San Diego, CA). CHO/NZ-12 cells were
cultivated in CHO-S-SFM II medium (Thermo Fisher Scientific, Inc.). NZ-12 was purified using Protein G-Sepharose(GE healthcare Bio-Sciences, Pittsburgh, PA). Human IgG
was purchased from Sigma-Aldrich Corp. (St. Louis, MO).
Determination of the binding affinity by flow cytometry

LN319 cells (2 · 105 cells) were resuspended with 100 mLof serially diluted antibodies (NZ-1, NZ-8, or NZ-12; 0.02–
100 mg/mL) followed by FITC-labeled anti-human IgG or
Oregon green-labeled anti-rat IgG (Thermo Fisher Scientific,Inc.). Fluorescence data were collected using a cell analyzer(EC800; Sony Corp.). The dissociation constants (KD) were

KANEKO ET AL.

obtained by fitting the binding isotherms using the built-in
one-site binding models in Prism software.
Preparation of effector cells

The preparation methods of effector cells have been described previously.(31) Human peripheral blood mononuculear cells (MNCs) were obtained from leukocytes, whichwere separated from peripheral blood of healthy donors.
The human study was approved by the Ethics Committee ofTokushima University.
Antibody-dependent cellular cytotoxicity

ADCC was determined with 51Cr release assay.(31,32)
Target cells were incubated with 0.1 mCi of 51Cr-sodium
chromate at 37C for 1 hour. After washing with CRPMI1640(RPMI1640 medium including 10% FBS) three times, 51Crlabeled target cells were placed in 96-well plates in triplicate.
Effector cells and antibodies were added to the plates. After
6 hours incubation, 51Cr release of the supernatant from eachwell (100 mL) was measured using a gamma counter (PerkinElmer, Waltham, MA). The percentage of cytotoxicity wascalculated 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.
Complement-dependent cytotoxicity

CDC was evaluated by 51Cr release assay as described
previously.(31,32) Target cells were incubated with 51Crsodium chromate (0.1 mCi) for 1 hour at 37C. After that, thecells were washed with CRPMI1640. 51Cr-labeled cells
were incubated with baby rabbit complement (Cedarlane,
Ontario, Canada) at a dilution of 1:4 and antibodies (1 mg/mL) for 6 hours in 96-well plates. After incubation, thesupernatant including 51Cr was measured using a gamma
counter. The percentage of cytotoxicity was calculated as
described above.
Statistical analyses

The statistical significance of differences in in vitro data
was analyzed by standard Student’s t-test. In this study,
p-values less than 0.05 were considered significant in all
experiments.
Results and Discussion
Development of a novel human–rat chimericanti-podoplanin antibody (NZ-12)

The sensitivity of NZ-8 is lower than that of NZ-1.(29) NZ8 was generated by fusing the VH of rat antibody (NZ-1) with
CH of human IgG1 and fusing VL regions of NZ-1 with CL ofhuman kappa because almost all chimeric antibodies and humanized antibodies are comprised of kappa light chains.(33,34)
In contrast, NZ-1 possesses lambda light chain, the rare isotype
found among rat IgGs (<5%).(35) Therefore, we subsequently
generated the human–rat chimeric antibody (NZ-12) by fusing
the VH of rat antibody (NZ-1) with CH of human IgG1 andfusing VL regions of NZ-1 with CL of human lambda.
NZ-12 demonstrated high sensitivity against podoplaninexpressing cancer cell lines in flow cytometry, and its reactivity was shown to be much higher than that of NZ-8 (dataCHIMERIC ANTI-HUMAN PODOPLANIN NZ-12

not shown). KD of NZ-1 was determined to be 1.2 · 10-9 M byflow cytometry (Fig. 1). Using the same methods, KD of NZ-8
was determined to be 7.3 · 10-7 M (Fig. 1), indicating that thebinding affinity of NZ-8 was diminished following conversion from rat mAb to a human–rat chimeric antibody. In
contrast, KD of NZ-12 was determined to be 8.5 · 10-9 M byflow cytometry (Fig. 1), indicating that the binding affinity
against a podoplanin-expressing cell line (LN319) becamemuch higher following conversion from a human kappa lightchain (NZ-8) to a human lambda light chain (NZ-12).

FIG. 1. Determination of binding-affinity using flow cytometry. LN319 (2 · 105 cells) was resuspended at 100 mL ofserially diluted NZ-1, NZ-8, or NZ-12 (0.02–100 mg/mL).

27
ADCC and CDC mediated by anti-podoplaninantibodies

To apply targeted therapy to podoplanin, we further assessed whether anti-podoplanin antibodies could induce
ADCC against podoplanin-expressing cell lines mediated byhuman MNC as effector cells. We compared the ADCC and
CDC activities against NZ-1, NZ-8, and NZ-12 using LN319,
D397, and PC-10 cell lines. ADCC against podoplaninexpressing cell lines was not exhibited by NZ-1 using human
MNC, whereas NZ-8 and NZ-12 showed the induction ofsignificant level of ADCC mediated by human MNCs against
LN319 and D397 glioblastoma cells and PC-10 lung cancer cells (Fig. 2A). Furthermore, ADCC by NZ-12 washigher compared with NZ-8 particularly against LN319 andD397 glioblastoma cells. Interestingly, CDC was induced by

FIG. 2. ADCC and CDC activities of anti-podoplaninantibodies. (A) ADCC activities induced by human MNC
against podoplanin-expressing cell lines LN319, D397, andPC-10 were determined with 6 hours 51Cr release assay at
the E/T ratio of 100 in the presence of 1 mg/mL of NZ-1,
NZ-8, NZ-12, and human IgG. (B) CDC activities against
podoplanin-expressing cell lines LN319, D397, and PC-10
were determined by 51Cr release assay. **p < 0.01, *p < 0.05
versus control (values are means – S.E.). ADCC, antibodydependent cellular cytotoxicity; CDC, complementdependent cytotoxicity; MNC, mononuculear cell.

28

KANEKO ET AL.

NZ-12 against all podoplanin-expressing cell lines, and thiswas significantly higher than that of NZ-8 (Fig. 2B).
Taken together, NZ-12 could be useful for antibody therapy against human podoplanin-expressing cancers. In the
future, NZ-1/NZ-12 could be further applied to the novelanti-tumor reagents, including T cells and viruses.(36,37)
Acknowledgments

We thank Miyuki Yanaka, Takuro Nakamura, Noriko
Saidoh, and Kanae Yoshida for their excellent technical assistance. This work was supported in part by the Basic Science and Platform Technology Program for Innovative
Biological Medicine from Japan Agency for Medical Research and development, AMED (Y.K.), by the Platform for
Drug Discovery, Informatics, and Structural Life Science(PDIS) from AMED (Y.K., T.M.), by the Project for utilizing glycans in the development of innovative drug discovery
technologies from AMED (Y.K.), by the Translational Research Network Program from AMED (Y.K.), by the Regional
Innovation Strategy Support Program from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT)
of Japan (Y.K.), by JSPS KAKENHI Grant Number 26440019(M.K.K.) and 16K10748 (Y.K.). This work was performed inpart under the Cooperative Research Program of Institute forProtein Research, Osaka University, CR-16-05 and by the
Grant for Joint Research Project of the Institute of Medical
Science, the University of Tokyo. The authors would like tothank Enago (www.enago.jp) for the English language review.
Author Disclosure Statement

No competing financial interests exist.
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Address correspondence to:
Dr. Yukinari Kato
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: November 17, 2016
Accepted: January 6, 2017