MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 5, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0030

Epitope Mapping of a Cancer-Specific Anti-Podocalyxin
Monoclonal Antibody (PcMab-60) Using Enzyme-Linked
Immunosorbent Assay and Surface Plasmon Resonance

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Teizo Asano,1 Hiroyuki Suzuki,2 Mika K. Kaneko,1 and Yukinari Kato1,2

Podocalyxin (PODXL) is a type I transmembrane sialoglycoprotein that is overexpressed in human cancers, including breast, oral, and lung. PODXL promotes tumor progression, and its expression is associated with poor
prognosis. Since PODXL is expressed in normal cells, including kidney podocytes and vascular endothelial cells(VECs), cancer-specific monoclonal antibodies (mAbs) are necessary to reduce the adverse effects of antibody
therapy on PODXL-expressing cancers. Previously, we established a cancer-specific mAb against PODXL, PcMab60 (mouse IgM, kappa), by immunizing mice with soluble PODXL produced by LN229 glioblastoma cells. PcMab60 reacted with PODXL-expressing cancer cells, but did not react with VECs. In this study, we investigated an
epitope of PcMab-60 using flow cytometry, surface plasmon resonance (SPR), and enzyme-linked immunosorbent
assay (ELISA). The results of SPR revealed that the PcMab-60 epitope consisted of Thr105, Arg109, Gly110,
Gly111, Gly112, Ser113, Gly114, Asn115, Pro116, and Thr117. In contrast, the results of ELISA revealed that the
PcMab-60 epitope consisted of Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, and Pro116. These
results demonstrate the cancer-specific epitope, which was recognized by PcMab-60.
Keywords: podocalyxin, PODXL, monoclonal antibody, epitope mapping, surface plasmon resonanceIntroduction

P

odocalyxin (PODXL) is a type I transmembrane sialoglycoprotein with a molecular weight of 150,000–
200,000,(1–6) PODXL was identified as an apical membraneprotein predominantly expressed in the kidney glomeruli.(7)PODXL consists of an extensively O-glycosylated and sialylated extracellular domain, a single-pass transmembrane
domain, and a highly conserved cytoplasmic domain with a
PDZ-binding region at the C-terminus.(8–10) PODXL is expressed on the surface of normal cells, including kidney podocytes, platelets, hematopoietic stem cells, and vascularendothelial cells.(7,11–13) PODXL is essential for maintaining
open filtration pathways between neighboring podocyte footprocesses through charge-repulsive effects of its highly
negatively charged extracellular domain.(7) PODXL is also
expressed in several human cancers, including breast,(14)oral,(15) and lung,(16,17) and plays crucial roles in tumor cellproliferation, invasion, and metastasis.(14,18)
Previously, we established a cancer-specific monoclonal
antibody (CasMab) technology against cell surface proteinsand developed some CasMabs, including anti-podoplanin
monoclonal antibody (mAb; LpMab-2)(19) Recently, we

successfully developed a novel CasMab against PODXL,
PcMab-60 (mouse IgM, kappa), by immunizing mice withthe cancer-derived soluble PODXL.(20) In that study, we
converted the mouse IgM subclass of PcMab-60 into a mouse
IgG2a subclass (60-mG2a) for antibody-dependent cellularcytotoxicity (ADCC). Then, we developed a core fucosedeficient type of 60-mG2a (60-mG2a-f) to potentiate the
ADCC activity of PcMab-60. We showed that 60-mG2a-f has
antitumor activity against a mouse xenograft of pancreaticcancer. The epitope of PcMab-60 has not been identified although the epitope identification of mAbs is essential to avoid
unexpected cross-reactivity and develop an antibody drug.
In this study, we clarified the critical epitope of PcMab-60
using enzyme-linked immunosorbent assay (ELISA), flow
cytometry, and surface plasmon resonance (SPR).
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 cells were purchasedfrom the American Type Culture Collection (ATCC, Manassas, VA, USA). PODXL mutant plasmids were transfected
into cells using Lipofectamine 2000 (Thermo Fisher

Departments of 1Antibody Drug Development and 2Molecular Pharmacology, Tohoku University Graduate School of Medicine,
Sendai, Japan.

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ASANO ET AL.

Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s instructions. Stable transfectants were sorted using a cell sorter (SH800; Sony Corp., Tokyo, Japan). Cells
were cultured in RPMI 1640 medium (Nacalai Tesque, Inc.,
Kyoto, Japan) supplemented with 10% heat-inactivated fetal
bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL
penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified
atmosphere containing 5% CO2. Transfectants were cultivated in a medium containing 0.5 mg/mL G418 (Nacalai
Tesque, Inc.).

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Plasmid preparation

cDNA encoding the full-length open reading frame of
PODXL was obtained through polymerase chain reaction(PCR) using cDNA derived from the LN229 cell line (ATCC)
as a template. We produced PODXL deletion mutants (dN60,
dN100, dN140, dN200, dN300, and dN400) using HotStar
HiFidelity polymerase kit (Qiagen Inc., Hilden, Germany)with appropriate oligonucleotides. In-Fusion PCR cloning kit(Takara Bio, Inc., Shiga, Japan) was used to subclone PCR
products into a pCAG-Neo vector (FUJIFILM Wako Pure
Chemical Industries Ltd., Osaka, Japan) containing a signal
sequence and PA tag (GVAMPGAEDDVV),(21) which isrecognized by an anti-PA tag mAb (NZ-1).(22) All amino acidnumbers were consistent with the NCBI reference sequence(NP_005388.2).

Table 2. Epitope Mapping of PcMab-60 by
Surface Plasmon Resonance Using
Alanine-Substituted Peptides
PeptideSequence

KD (nM)

100–119
S100A
G101A
P102A
V103A
N104A
T105A
T106A
V107A
A108G
R109A
G110A
G111A
G112A
S113A
G114A
N115A
P116A
T117A
T118A
T119A

SGPVNTTVARGGGSGNPTTTAGPVNTTVARGGGSGNPTTTSAPVNTTVARGGGSGNPTTTSGAVNTTVARGGGSGNPTTTSGPANTTVARGGGSGNPTTTSGPVATTVARGGGSGNPTTTSGPVNATVARGGGSGNPTTTSGPVNTAVARGGGSGNPTTTSGPVNTTAARGGGSGNPTTTSGPVNTTVGRGGGSGNPTTTSGPVNTTVAAGGGSGNPTTTSGPVNTTVARAGGSGNPTTTSGPVNTTVARGAGSGNPTTTSGPVNTTVARGGASGNPTTTSGPVNTTVARGGGAGNPTTTSGPVNTTVARGGGSANPTTTSGPVNTTVARGGGSGAPTTTSGPVNTTVARGGGSGNATTT
SGPVNTTVARGGGSGNPATTSGPVNTTVARGGGSGNPTATSGPVNTTVARGGGSGNPTTA

399
570.8
418.2
453
351.1
385.1
ND
348.6
315.1
545.5
ND
ND
ND
ND
ND
ND
ND
ND
ND
564.5
534.2

ND, not determined.

Enzyme-linked immunosorbent assay
Flow cytometry

Cells were harvested by briefly exposing them to 0.25%
trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing the cells with 0.1% bovine serum
albumin (BSA) in phosphate-buffered saline (PBS), they
were treated with PcMab-60 (10 lg/mL) or NZ-1 (1 lg/mL)
for 30 min at 4C and then with Alexa Fluor 488-conjugatedantimouse IgG or antirat IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA). Fluorescence data werecollected using BD FACSLyric system (Becton, Dickinson
and Company, Franklin Lakes, NJ, USA).
PODXL peptides

PODXL (Accession No. NP_005388.2) peptides, including 6 deletion mutants and 20 alanine-substituted peptides(Tables 1–3), were synthesized using PEPScreen (SigmaAldrich Corp., St. Louis, MO, USA).

Table 1. Epitope Mapping of PcMab-60 by
Enzyme-Linked Immunosorbent Assay Using
Deletion Mutants
PeptideSequence

PcMab-60

70–89
80–99
90–109100–119
110–129
120–139KANEILASVKATTLGVSSDSATTLGVSSDSPGTTTLAQQV
PGTTTLAQQVSGPVNTTVAR
SGPVNTTVARGGGSGNPTTT
GGGSGNPTTTIESPKSTKSAIESPKSTKSADTTTVATSTA+++-+++, OD655 ‡ 0.1; ++, OD655 ‡ 0.08; +, OD655 ‡ 0.06; -,OD655 < 0.06.

Synthesized PODXL peptides were immobilized in Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at a concentration of 10 lg/mL for 30 min at
37C. After washing with PBS containing 0.05% Tween 20(PBST) (Nacalai Tesque, Inc.), wells were blocked with 1%

Table 3. Epitope Mapping of PcMab-60 by
Enzyme-Linked Immunosorbent Assay Using
Alanine-Substituted Peptides
PeptideSequence

PcMab-60

S100A
G101A
P102A
V103A
N104A
T105A
T106A
V107A
A108G
R109A
G110A
G111A
G112A
S113A
G114A
N115A
P116A
T117A
T118A
T119A

AGPVNTTVARGGGSGNPTTTSAPVNTTVARGGGSGNPTTTSGAVNTTVARGGGSGNPTTTSGPANTTVARGGGSGNPTTTSGPVATTVARGGGSGNPTTTSGPVNATVARGGGSGNPTTTSGPVNTAVARGGGSGNPTTTSGPVNTTAARGGGSGNPTTTSGPVNTTVGRGGGSGNPTTTSGPVNTTVAAGGGSGNPTTTSGPVNTTVARAGGSGNPTTTSGPVNTTVARGAGSGNPTTTSGPVNTTVARGGASGNPTTTSGPVNTTVARGGGAGNPTTTSGPVNTTVARGGGSANPTTTSGPVNTTVARGGGSGAPTTTSGPVNTTVARGGGSGNATTT
SGPVNTTVARGGGSGNPATTSGPVNTTVARGGGSGNPTATSGPVNTTVARGGGSGNPTTA

+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+++
+++

+++, OD655 ‡ 0.1; ++, OD655 ‡ 0.08; +, OD655 ‡ 0.06; -,OD655 < 0.06.

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FIG. 1. Epitope mapping of PcMab-60 using deletion mutants. (A, B) PODXL deletion mutants were expressed in CHOK1 cells, incubated with PcMab-60 (A) or anti-PA tag mAb (NZ-1) (B) for 30 min at 4C, and analyzed using flow
cytometry, followed by corresponding secondary antibody treatment. Lines, with first antibody; shadowed, without firstantibody in blocking buffer (negative control). (C) Schematic illustration of PcMab-60 epitope mapping with PODXL
deletion mutants (dN60, dN100, dN140, dN200, dN300, and dN400) with a PA tag at N-terminus, and synthesized peptides
corresponding to 70–89, 80–99, 90–109, 100–119, 110–129, and 120–139 amino acids. Black bars: positive reaction to
PcMab-60. White bars: negative reaction to PcMab-60. IC, intracellular domain; PA, PA tag; PODXL, podocalyxin; TM,
transmembrane domain.

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BSA-containing PBST for 30 min at 37C. The plates wereincubated with PcMab-60 (20 lg/mL), followed by aperoxidase-conjugated antimouse immunoglobulins (1:2000
diluted; Agilent Technologies, Inc., Santa Clara, CA, USA).
Enzymatic reactions were conducted using ELISA POD
Substrate TMB kit (Nacalai Tesque, Inc.). Optical density
was measured at 655 nm using iMark microplate reader (BioRad Laboratories, Inc., Berkeley, CA, USA).

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Measurement of dissociation constants (KD)between PcMab-60 and alanine-substituted
peptides using SPR

PcMab-60 was immobilized on the sensor chip CM5 according to the protocol described by Cytiva (Marlborough,MA, USA). In brief, PcMab-60 was diluted to 10 lg/mLusing acetate buffer (pH 4.0: Cytiva) and immobilized using
an amine-coupling reaction. The surface of the flow cell 2 ofthe sensor chip CM5 was treated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (NHS),
followed by injecting PcMab-60. Unreacted NHS-ester was
blocked with ethanolamine after PcMab-60 immobilization. KD values between PcMab-60 and alanine-substituted
peptides given in Table 2 were measured using Biacore
X100 (Cytiva) at 25C. The buffer used was PBS containing 0.05% (v/v) Tween 20 and 1.4% (v/v) dimethyl
sulfoxide (FUJIFILM Wako Pure Chemical Corporation).
A single cycle kinetics method was used to measure binding signals. We conducted an equilibrium analysis to analyze the data and determine KD using BIAevaluation
software (Cytiva).

ASANO ET AL.
Results
Determination of the PcMab-60 epitope using PODXL
deletion mutants and synthesized peptides

To determine the PcMab-60 epitope, we constructed
PODXL N-terminal deletion mutants (dN60, dN100, dN140,
dN200, dN300, and dN400) with the PA tag at theirN-terminus and expressed these mutants in CHO-K1 cells.
Reactions between PcMab-60 and deletion mutants and between anti-PA tag mAb (NZ-1) and deletion mutants werecharacterized using flow cytometry. The results of flow cytometry showed that PcMab-60 reacted with dN60 and
dN100, but did not react with dN140, dN200, dN300, anddN400 (Fig. 1A). All deletion mutants were detected by NZ-1
(Fig. 1B). Both PcMab-60 and NZ-1 did not recognize parental CHO-K1 cells (Fig. 1A, B). These results indicated that
the N-terminus of PcMab-60 epitope existed between the
100–140 amino acids (aa) of PODXL (Fig. 1C).
Next, we prepared six peptides of 20 aa each (70–89, 80–
99, 90–109, 100–119, 110–129, and 120–139 of PODXL;Table 1) and performed ELISA. PcMab-60 reacted with the
100–119 peptide (100-SGPVNTTVARGGGSGNPTTT-119),but did not react with other peptides (Table 1). This result
indicated that the PcMab-60 epitope exists between the 100–
119 aa of PODXL (Fig. 1C).
Epitope mapping using SPR

Next, we synthesized wild-type and 20 alanine-substituted
PODXL peptides of 100–119 aa and determined the KD
values using SPR. The KD values between 11 peptides

FIG. 2. Schematic illustration of PcMab-60 epitope mapping using SPR (A) and ELISA (B). Critical epitope residues areunderlined. ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance; SS, signal sequence.

EPITOPE MAPPING OF PCMAB-60(S100A, G101A, P102A, V103A, N104A, T106A, V107A,
A108G, T118A, T119A, and wild-type 100–119) and
PcMab-60 were determined to be *300–600 nM. However,the KD values between 10 peptides (T105A, R109A, G110A,
G111A, G112A, S113A, G114A, N115A, P116A, andT117A) and PcMab-60 were not determined. The KD values
for each peptide are given in Table 2. These results indicatedthat Thr105, Arg109, Gly110, Gly111, Gly112, Ser113,
Gly114, Asn115, Pro116, and Thr117 are the critical epitopeof PcMab-60 (Fig. 2A).

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Epitope mapping using ELISA

We further evaluated the reactivity of PcMab-60 to thealanine-substituted PODXL peptides using ELISA. PcMab60 reacted with S100A, G101A, P102A, V103A, N104A,
T105A, T106A, V107A, A108G, T117A, T118A, and
T119A, but not with R109A, G110A, G111A, G112A,
S113A, G114A, N115A, and P116A (Table 3). The results of
ELISA indicated that Arg109, Gly110, Gly111, Gly112,
Ser113, Gly114, Asn115, and Pro116 are the critical epitopeof PcMab-60 (Fig. 2B).
Discussion

In this study, we determined the epitope of PcMab-60
using flow cytometry, SPR, and ELISA. We determined
Thr105, Arg109, Gly110, Gly111, Gly112, Ser113,
Gly114, Asn115, Pro116, and Thr117 of PODXL as thecritical epitope of PcMab-60 using SPR and Arg109,
Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, andPro116 of PODXL as the critical epitope of PcMab-60
using ELISA. The epitope from Arg109 to Pro116 iscommon in both ELISA and SPR. In contrast, Thr105 andThr117 are additionally determined in SPR analysis.
PcMab-60 was immobilized on a sensor chip in SPR
analysis. In contrast, peptides were immobilized on immunoplates in ELISA. Immobilized molecules were different between ELISA and SPR, which may influence thereactivity of PcMab-60 to these peptides.
In our previous study, we successfully developed antipodoplanin CasMabs, including LpMab-2, which specificallyrecognizes cancer-type podoplanin, but not normal-type podoplanin.(19) LpMab-2 recognized both the Thr55-Leu64
peptide of human podoplanin and cancer-type aberrant glycosylation of Thr55 and/or Ser56, which is well glycosylated
in glioblastoma cells.(19) PcMab-60 reacted with PODXLexpressing pancreatic cancer cells, but not with normal
cells.(20) In this study, SPR analysis revealed that the PcMab60 epitope includes Thr105, Ser113, Asn115, and Thr117,
which is different from the epitope recognized by previously
established anti-PODXL mAb, PcMab-47 (mouse IgG1,
kappa). PcMab-47 reacts with Asp207, His208, Leu209, andMet210 and recognizes both normal and cancer-type
PODXL.(23) Therefore, the PcMab-60 epitope can be modified by O- and/or N-glycosylation in cells. In this study, weused synthesized peptides (no glycosylation) in SPR and
ELISA analysis, which may influence the reactivity of
PcMab-60. Further analyses of glycosylation at these sites
and of the difference between normal and cancer cells areneeded to understand the cancer-specific recognition to
PODXL by PcMab-60.

231
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) under Grant
Numbers JP21am0401013 (Y.K.), JP21am0101078 (Y.K.),
and JP21bm1004001 (Y.K.), and by the Japan Society for the
Promotion of Science ( JSPS) Grants-in-Aid for Scientific
Research (KAKENHI) Grant Numbers 21K15523 (to T.A.),
21K07168 (to M.K.K.), and 19K07705 (to Y.K.).
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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: June 27, 2021
Accepted: September 1, 2021