Biochemistry and Biophysics Reports 14 (2018) 78–82

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Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

Determination of critical epitope of PcMab-47 against human podocalyxinaaa

Shunsuke Itai , Shinji Yamada , Mika K. Kaneko , Yukinari Katoaba,b,⁎

T

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapanNew Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapanA R T I C LE I N FO

A B S T R A C T

Keywords:
Podocalyxin
PODXL
Epitope mapping
Monoclonal antibody
Oral cancer

Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed insome types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor
growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, ﬂow cytometry, and immunohistochemical analysis. In thisstudy, we used enzyme-linked immunosorbent assay (ELISA), ﬂow cytometry, and immunohistochemical
analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207,His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized
PcMab-47 reaction against oral cancer cells by ﬂow cytometry and immunohistochemical analysis. Theseﬁndings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for
antitumor activities.

1. Introduction

47-mG2a-f exerted antitumor activities in HSC-2 xenograft models at adose of 500 μg/mouse/week administered twice, 47-mG2a-f showed ahigher antitumor activity than 47-mG2a. These results suggested that acore fucose-deﬁcient anti-PODXL mAb could be useful for antibodybased therapy against PODXL-expressing OSCCs. Although engineeredmAbs of PcMab-47 show high antitumor activities against cancer cells,
the critical epitope of PcMab-47 remains to be identiﬁed.
In this study, we clariﬁed the binding epitope of PcMab-47 using
enzyme-linked immunosorbent assay (ELISA), ﬂow cytometry, andimmunohistochemistry.

Podocalyxin (PODXL) is a type I transmembrane protein, which ishighly glycosylated and has a molecular weight of 150,000–200,000[1–5]. PODXL expression has been reported in several human cancers,
including oral [6], breast [7], and lung cancer [8,9] and leads to tumor
growth, invasion, and metastasis [7,10]. Several studies have developed
anti-PODXL monoclonal antibodies (mAbs) [11,12].
We previously produced a novel anti-PODXL mAb, PcMab-47 (IgG1,kappa) [13]. PcMab-47 reacts with endogenous PODXL-expressing
cancer cell lines and normal cells independently of glycosylation inWestern blot, ﬂow cytometry, and immunohistochemical analysis. We
engineered PcMab-47 into 47-mG2a, a mouse IgG2a-type mAb, to add
antibody-dependent cellular cytotoxicity (ADCC). We further developed
47-mG2a-f, a core fucose-deﬁcient type of 47-mG2a, to augment its
ADCC. Immunohistochemical analysis of oral cancer tissues using
PcMab-47 and 47-mG2a revealed that the latter stained oral squamouscell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower
concentration than the former. In vitro analysis showed that 47-mG2a-f
exhibited a much stronger ADCC than 47-mG2a against OSCC cells. In
vivo analysis revealed that 47-mG2a-f, but not 47-mG2a, exerted anantitumor activity in SAS and HSC-2 xenograft models at a dose of
100 μg/mouse/week administered three times. Although 47-mG2a and

2. Materials and methods
2.1. Cell lines
CHO-K1 was obtained from the American Type Culture Collection(ATCC, Manassas, VA). SAS (oral squamous carcinoma cell line fromtongue) was obtained from the Japanese Collection of Research
Bioresources Cell Bank (Osaka, Japan). CHO-K1 cells were transfectedwith PA-tagged PODXL deletion mutant plasmids using Lipofectamine
LTX (Thermo Fisher Scientiﬁc Inc., Waltham, MA). PODXL deletionmutants were cultured in an RPMI 1640 medium (Nacalai Tesque, Inc.,Kyoto, Japan), and SAS was cultured in Dulbecco's Modiﬁed Eagle'sAbbreviations: mAb, monoclonal antibody; SCC, squamous cell carcinoma; DMEM, Dulbecco's Modiﬁed Eagle's Medium; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum
albumin; PBS, phosphate-buﬀered saline; FBS, fetal bovine serum; DAB, 3,3-diaminobenzidine tetrahydrochloride⁎Corresponding author at: New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
E-mail addresses: yukinarikato@med.tohoku.ac.jp, yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2018.04.003
Received 17 March 2018; Accepted 9 April 2018
2405-5808/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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Fig. 1. (A) Flow cytometry using deletion mutants of PODXL. PcMab-47 and anti-PA tag (NZ-1) were treated with deletion mutants of PODXL cells for 30 min at 4 °C,
followed by the addition of secondary antibodies. Gray peak, negative control; blue peak, anti-PA tag; red peak, PcMab-47. (B) Schematic illustration of the PcMab47-epitope.

2.2. Plasmid preparation

Medium (DMEM; Nacalai Tesque, Inc.), supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientiﬁc Inc.), 100
units/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml amphotericin B (Nacalai Tesque, Inc.), and incubated at 37 °C in a humidiﬁed
atmosphere of 5% CO2 and 95% air.

The cDNA encoding the full-length open reading frame (ORF) ofPODXL was obtained by PCR using cDNA derived from the LN229 cellline (ATCC) as a template. Appropriate oligonucleotides were used as79

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primers to make each deletion mutants. PCR products were subcloned
into pCAG vector (FUJIFILM Wako Pure Chemical Industries Ltd.,Osaka, Japan) with signal sequence and PA tag using the In-Fusion PCR
Cloning kit (Takara Bio, Inc., Shiga, Japan). All amino acid numberwere consistent with the NCBI Reference Sequence, NP_005388.2.

Table 1
Determination of PcMab-47 Epitope by ELISA.

2.3. Enzyme-linked immunosorbent assay (ELISA)
Synthesized PODXL peptides (PEPScreen; Sigma-Aldrich Corp., St.
Louis, MO) were immobilized on Nunc Maxisorp 96-well immunoplates(Thermo Fisher Scientiﬁc Inc.) at 1 μg/ml for 30 min. After blocking
with SuperBlock T20 (PBS) Blocking Buﬀer (Thermo Fisher ScientiﬁcInc.), the plates were incubated with puriﬁed PcMab-47 (1 μg/ml),
followed by a 1:2000 dilution of peroxidase-conjugated anti-mouse IgG(Agilent Technologies Inc., Santa Clara, CA). The enzymatic reaction
was conducted using 1-Step Ultra TMB-ELISA (Thermo Fisher ScientiﬁcInc.). Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA). These reactions
were performed at 37 °C with a total sample volume of 50–100 μl.
2.4. Flow cytometry
Cells were harvested after brief exposure to 0.25% trypsin/1 mM
ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). Afterwashing with 0.1% bovine serum albumin in PBS, the cells were treatedwith PcMab-47 (1 μg/ml) or PcMab-47 (1 μg/ml) plus peptides (1 μg/ml) for 30 min at 4 °C, followed by treatment with Alexa Fluor 488conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc.,Danvers, MA). Fluorescence data were acquired using the Cell Analyzer
SA3800 (Sony Corp., Tokyo, Japan).

PeptideSequence

PcMab-47

180–199
190–209
200–219
210–229
220–239
230–249
T200A
P201A
T202A
S203A
S204A
G205A
H206A
D207A
H208A
L209A
M210A
K211A
I212A
S213A
S214AS215AS216AS217AT218AV219ATPHPTSPLSPRQPTSTHPVARQPTSTHPVATPTSSGHDHLTPTSSGHDHLMKISSSSSTVMKISSSSSTVAIPGYTFTSPAIPGYTFTSPGMTTTLPSSVGMTTTLPSSVISQRTQQTSSAPTSSGHDHLMKISSSSSTVTATSSGHDHLMKISSSSSTVTPASSGHDHLMKISSSSSTVTPTASGHDHLMKISSSSSTVTPTSAGHDHLMKISSSSSTVTPTSSAHDHLMKISSSSSTVTPTSSGADHLMKISSSSSTVTPTSSGHAHLMKISSSSSTVTPTSSGHDALMKISSSSSTVTPTSSGHDHAMKISSSSSTVTPTSSGHDHLAKISSSSSTVTPTSSGHDHLMAISSSSSTVTPTSSGHDHLMKASSSSSTVTPTSSGHDHLMKIASSSSTVTPTSSGHDHLMKISASSSTVTPTSSGHDHLMKISSASSTVTPTSSGHDHLMKISSSASTVTPTSSGHDHLMKISSSSATVTPTSSGHDHLMKISSSSSAVTPTSSGHDHLMKISSSSSTA−−＋＋＋−−−＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋−−−−＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋＋, OD655 ≧ 0.7; ＋＋, 0.4 ≦ OD655＜0.7; ＋, 0.1 ≦ OD655＜0.4; -, OD655＜0.1.

deletion mutants of PODXL, including dN25, dN80, dN100, dN140,dN180, dN200, dN220, and dN300 (Fig. 1A, upper panel). In contrast,
PcMab-47 did not react with dN220 and dN300 (Fig. 1A, lower panel).
These results indicate that the N-terminus of PcMab-47 epitope exists
between the 200th and 219th aa. These results are summarized inFig. 1B.
We next synthesized a series of PODXL peptides such as 180–199 aa,
190–209 aa, 200–219 aa, 210–229 aa, 220–239 aa, and 230–249 aa(Table 1, Fig. 1B). PcMab-47 reacted only with 200–219 aa peptides in
ELISA. We synthesized a series of point mutations of PODXL peptides of200–219 aa (Table 1). PcMab-47 reacted with T200A, P201A, T202A,S203A, S204A, G205A, H206A, K211A, S213A, S214A, S215A, S216A,S217A, T218A, and V219A. PcMab-47 weakly reacted with I212A anddid not react with D207A, H208A, L209A, or M210A, indicating that
PODXL peptides from the 207th to the 210th aa (207-DHLM−210 sequence) constitute the critical minimum epitope of PcMab-47.
We further performed a blocking assay using ﬂow cytometry.
PcMab-47 reacted with the SAS cell line (Fig. 2). This reaction wascompletely neutralized by the wild-type peptide (200-TPTSSGHDHLMKISSSSSTV-219), H206A, and K211A. In contrast, D207A, H208A,
L209A, and M210A did not block the reaction of PcMab-47 with SAS,
conﬁrming that the (207-DHLM-210) sequence is a critical epitope ofPcMab-47.
Finally, we performed a blocking assay using immunohistochemistry. PcMab-47 reacted with oral cancer tissues(Fig. 3A) and the reaction was completely neutralized by the wild-type
peptide (200-TPTSSGHDHLMKISSSSSTV-219) and H206A. D207A did notblock the reaction of PcMab-47, and this conﬁrmed the results of theﬂow cytometric blocking assays.
Taken together, our results indicate that the critical epitope ofPcMab-47 is Asp207, His208, Leu209, and Met210. Our ﬁndings could
lead to the production of more functional anti-PODXL mAbs, which
would be advantageous for antitumor eﬀects against PODXL-expressing
cancers.

2.5. Immunohistochemical analyses
Histological sections of 4-μm thickness were directly autoclaved incitrate buﬀer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for
20 min. Sections were then incubated with PcMab-47 (5 μg/ml) or
PcMab-47 (5 μg/ml) plus peptides (5 μg/ml) for 1 h at room temperature, treated using an Envision+ kit (Agilent Technologies Inc.) for
30 min. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min, and counterstained with hematoxylin (FUJIFILM Wako Pure Chemical IndustriesLtd.).
3. Results and discussion
We previously developed PcMab-47, a novel anti-PODXL mAb
which exhibits a high speciﬁcity and sensitivity against human PODXL.
PcMab-47 was shown to be useful for immunohistochemical analyses
using paraﬃn-embedded tissues [13,14]. Furthermore, engineeredmAbs of PcMab-47 exerted high antitumor activities against cancer
cells in mouse xenograft models. Therefore, the determination of thebinding epitope of PcMab-47 is critical to further develop a moleculartargeting therapy against PODXL.
In this study, eight deletion mutants of PODXL were constructed(Fig. 1A). Stable transfections of PODXL-mutant clones were established on CHO-K1 cells, including dN25 (corresponding to 25–526
amino acids [aa]), dN80 (corresponding to 80–526 aa), dN100 (corresponding to 100–526 aa), dN140 (corresponding to 140–526 aa),dN180 (corresponding to 180–526 aa), dN200 (corresponding to200–526 aa), dN220 (corresponding to 220–526 aa), and dN300 (corresponding to 300–526 aa). All deletion mutants of PODXL contained
an N-terminal PA tag and were analyzed using ﬂow cytometry for theepitope mapping of PcMab-47. NZ-1 (anti-PA tag mAb) detected all80

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Fig. 2. (A) Flow cytometry using SAS cells. PcMab-47 (1 μg/ml) and PcMab-47 (1 μg/ml) + wild-type peptide (200-TPTSSGHDHLMKISSSSSTV-219; 1 μg/ml) weretreated with SAS cells for 30 min at 4 °C, followed by the addition of secondary antibodies. Gray peak, negative control; red peak, PcMab-47. (B) Flow cytometryusing SAS cells. PcMab-47 (1 μg/ml) + each peptide (1 μg/ml) was treated with SAS cells for 30 min at 4 °C, followed by the addition of secondary antibodies. Graypeak, negative control; red peak, PcMab-47 + each peptide.

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(Y.K.), and by JSPS KAKENHI Grant no. 17K07299 (M.K.K.) and Grant
no. 16K10748 (Y.K.).
Appendix A. Transparency document
Transparency document associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.bbrep.2018.04.003.
References[1] K.M. McNagny, I. Pettersson, F. Rossi, I. Flamme, A. Shevchenko, M. Mann, T. Graf,Thrombomucin, a novel cell surface protein that deﬁnes thrombocytes and multipotent hematopoietic progenitors, J. Cell Biol. 138 (1997) 1395–1407.
[2] W.M. Schopperle, D.B. Kershaw, W.C. DeWolf, Human embryonal carcinoma tumor
antigen, Gp200/GCTM-2, is podocalyxin, Biochem. Biophys. Res. Commun. 300(2003) 285–290.
[3] W.M. Schopperle, W.C. DeWolf, The TRA-1-60 and TRA-1-81 human pluripotent
stem cell markers are expressed on podocalyxin in embryonal carcinoma, Stem Cells
25 (2007) 723–730.
[4] D. Kerjaschki, H. Poczewski, G. Dekan, R. Horvat, E. Balzar, N. Kraft, R.C. Atkins,Identiﬁcation of a major sialoprotein in the glycocalyx of human visceral glomerular epithelial cells, J. Clin. Investig. 78 (1986) 1142–1149.
[5] D.B. Kershaw, S.G. Beck, B.L. Wharram, J.E. Wiggins, M. Goyal, P.E. Thomas,R.C. Wiggins, Molecular cloning and characterization of human podocalyxin-likeprotein. Orthologous relationship to rabbit PCLP1 and rat podocalyxin, J. Biol.
Chem. 272 (1997) 15708–15714.
[6] C.W. Lin, M.S. Sun, H.C. Wu, Podocalyxin-like 1 is associated with tumor aggressiveness and metastatic gene expression in human oral squamous cell carcinoma,Int. J. Oncol. 45 (2014) 710–718.
[7] K.A. Snyder, M.R. Hughes, B. Hedberg, J. Brandon, D.C. Hernaez, P. Bergqvist,
F. Cruz, K. Po, M.L. Graves, M.E. Turvey, J.S. Nielsen, J.A. Wilkins, S.R. McColl,J.S. Babcook, C.D. Roskelley, K.M. McNagny, Podocalyxin enhances breast tumor
growth and metastasis and is a target for monoclonal antibody therapy, BreastCancer Res. 17 (2015) 46.
[8] L.K. Koch, H. Zhou, J. Ellinger, K. Biermann, T. Holler, A. von Rucker, R. Buttner,I. Gutgemann, Stem cell marker expression in small cell lung carcinoma and developing lung tissue, Hum. Pathol. 39 (2008) 1597–1605.
[9] T. Tschernig, R. Pabst, M. Kasper, M. El-Hadi, B. Singh, Expression of caveolin-1 andpodocalyxin in rat lungs challenged with 2-kDa macrophage-activating lipopeptide
and Flt3L, Cell Tissue Res. 356 (2014) 207–216.
[10] T.N. Flores-Tellez, T.V. Lopez, V.R. Vasquez Garzon, S. Villa-Trevino, Co-Expressionof Ezrin-CLIC5-podocalyxin is associated with migration and invasiveness in hepatocellular carcinoma, PLoS One 10 (2015) e0131605.
[11] L. Kang, C. Yao, A. Khodadadi-Jamayran, W. Xu, R. Zhang, N.S. Banerjee,C.W. Chang, L.T. Chow, T. Townes, K. Hu, The universal 3D3 antibody of human
PODXL is pluripotent cytotoxic, and identiﬁes a residual population after extended
diﬀerentiation of pluripotent stem cells, Stem Cells Dev. 25 (2016) 556–568.
[12] H. Toyoda, Y. Nagai, A. Kojima, A. Kinoshita-Toyoda, Podocalyxin as a major
pluripotent marker and novel keratan sulfate proteoglycan in human embryonicand induced pluripotent stem cells, Glycoconj. J. 34 (2017) 139–145.
[13] S. Ogasawara, M.K. Kaneko, S. Yamada, R. Honma, T. Nakamura, N. Saidoh,
M. Yanaka, K. Yoshida, Y. Fujii, Y. Kato, PcMab-47: novel antihuman podocalyxin
monoclonal antibody for immunohistochemistry, Monoclon Antib. Immunodiagn.
Immunother. 36 (2017) 50–56.
[14] S. Itai, S. Yamada, M.K. Kaneko, H. Harada, Y. Kato, Immunohistochemical analysis
using antipodocalyxin monoclonal antibody PcMab-47 demonstrates podocalyxin
expression in oral squamous cell carcinomas, Monoclon Antib. Immunodiagn.
Immunother. 36 (2017) 220–223.

Fig. 3. (A) Immunohistochemistry using oral cancers. Oral cancer tissues wereautoclaved in a citrate buﬀer for 20 min. Sections were then incubated withPcMab-47 (5 μg/ml), PcMab-47 (5 μg/ml), + wild-type peptide (WT, 200-TPTSSGHDHLMKISSSSSTV-219; 5 μg/ml), PcMab-47 (5 μg/ml) + H206A (5 μg/ml),or PcMab-47 (5 μg/ml), + D207A (5 μg/ml) and treated using an Envision +kit. Color development was performed using 3,3-diaminobenzidine tetrahydrochloride. Sections were then counterstained with hematoxylin. The scale
bar represents 100 µm. (B) Schematic illustration of PODXL and the PcMab-47epitope. The critical epitope of PcMab-47 is 207-DHLM-210. SS, signal peptide;TM, transmembrane; IC, intracellular region.

Acknowledgments
We thank Takuro Nakamura, Miyuki Yanaka, Saori Handa, andYoshimi Nakamura for excellent technical assistance.
Funding
This research was supported in part by AMED under Grant nos.:
JP17am0301010 (Y.K.), JP17am0101078 (Y.K.), and JP17ae0101028

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