Biochemistry and Biophysics Reports 24 (2020) 100826

Contents lists available at ScienceDirect

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

A cancer-specific anti-podocalyxin monoclonal antibody (60-mG2a-f) exerts
antitumor effects in mouse xenograft models of pancreatic carcinoma
Mika K. Kaneko a, Tomokazu Ohishi b, Manabu Kawada b, Yukinari Kato a, c, *a

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, 18-24 Miyamoto, Numazu-shi, Shizuoka, 410-0301, Japanc
New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Podocalyxin
PODXL
Monoclonal antibody
Cancer-specific mAb
Pancreatic cancer

Overexpression of podocalyxin (PODXL) is associated with progression, metastasis, and poor outcomes in several
cancers. PODXL also plays an important role in the development of normal tissues. For antibody-based therapy totarget PODXL-expressing cancers using monoclonal antibodies (mAbs), cancer-specificity is necessary to reduce
the risk of adverse effects to normal tissues. In this study, we developed an anti-PODXL cancer-specific mAb(CasMab), named as PcMab-60 (IgM, kappa) by immunizing mice with soluble PODXL, which is overexpressed in
LN229 glioblastoma cells. The PcMab-60 reacted with the PODXL-overexpressing LN229 (LN229/PODXL) cells
and MIA PaCa-2 pancreatic cancer cells in flow cytometry but did not react with normal vascular endothelial
cells (VECs), whereas one of non-CasMabs, PcMab-47 showed high reactivity for not only LN229/PODXL and
MIA PaCa-2 cells but also VECs, indicating that PcMab-60 is a CasMab. Next, we engineered PcMab-60 into amouse IgG2a-type mAb, named as 60-mG2a, to add antibody-dependent cellular cytotoxicity (ADCC). We further
developed a core fucose-deficient type of 60-mG2a, named as 60-mG2a-f, to augment its ADCC activity. In vivo
analysis revealed that 60-mG2a-f exerted antitumor activity in MIA PaCa-2 xenograft models at a dose of 100 μg/
mouse/week administered three times. These results suggested that 60-mG2a-f could be useful for antibody-based
therapy against PODXL-expressing pancreatic cancers.

1. Introduction

recombinant soluble PODXL, which was purified from the culture su­
pernatant of LN229/ectodomain-PODXL cells, and developed PcMab-47
of mouse IgG1 subclass [17]. We then produced a mouse-humanchimeric mAb (chPcMab-47) and demonstrated its antitumor activity
against colorectal cancers [18]. Furthermore, we established a chimericanti-PODXL mAb (47-mG2a) by combining the variable region of
PcMab-47 of mouse IgG1 and the constant region of mouse IgG2a [19].
Moreover, we produced 47-mG2a-f, a core fucose-deficient 47-mG2a toaugment antibody-dependent cellular cytotoxicity (ADCC), anddemonstrated high antitumor activity of 47-mG2a-f against oral cancers[19]. Although PcMab-47 and its modified mAbs are very useful for
detecting PODXL in immunohistochemistry, they reacted with bothcancer cells and normal cells, including vascular endothelial cells[19–22]. Therefore, we could not transfer PcMab-47 into clinical use
because its chimeric mAb or humanized mAb might show severe adverse
effects.

Podocalyxin (PODXL), also known as TRA-1-60 and TRA-1-81 anti­
gens, is a highly N- or O-glycosylated type I transmembrane protein witha molecular weight of 150,000–200,000 [1–4], and is used as pluripo­
tent stem cell markers [3,5–9]. PODXL plays an important biologicalrole in the kidney, heart, pancreatic, and breast tissues [10], and is also aprognostic indicator and a diagnostic marker for several malignant tu­
mors such oral cancer [11], brain tumors [4], colorectal cancer [12], andrenal cancer [13]. Because PODXL has been suggested to promote tumor
growth, invasion, and metastasis [14,15], high PODXL expression could
have adverse effects on overall survival (OS), disease-specific survival(DSS), and disease-free survival (DFS) in several cancers.
Despite the development of anti-PODXL monoclonal antibodies(mAbs), the efficacy of these treatments against cancers remains to be
fully explained [5,16]. We previously immunized mice with

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; BSA, bovine serum albumin; CasMab, cancer-specific monoclonal antibody; CBIS, Cell-Based
Immunization and Screening; CDC, complement-dependent cytotoxicity; PBS, phosphate-buffered saline; PODXL, podocalyxin.
* Corresponding author. New Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
E-mail address: yukinarikato@med.tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2020.100826
Received 25 July 2020; Received in revised form 22 September 2020; Accepted 7 October 2020
2405-5808/© 2020 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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Biochemistry and Biophysics Reports 24 (2020) 100826

For antibody-based therapy to target PODXL-expressing cancers,
cancer-specificity is necessary to reduce the risk of adverse effects tonormal tissues. In this study, we developed a cancer-specific anti-PODXL
mAb, PcMab-60 (IgM, kappa) by immunizing mice with soluble PODXL.
We engineered PcMab-60 into a mouse IgG2a-type mAb (60-mG2a) to
add ADCC, and further produced a core fucose-deficient type of 60-mG2a(60-mG2a-f) to augment its ADCC activity. We then examined the anti­
tumor activity of 60-mG2a-f against a mouse xenograft of pancreaticcancer.

2.4. ELISA
Proteins were immobilized on Nunc Maxisorp 96-well immuno
plates (Thermo Fisher Scientific, Inc.) at 1 μg/mL for 30 min. After
blocking with 1% bovine-serum albumin (BSA) in 0.05% Tween 20/
phosphate buffered saline (PBS, Nacalai Tesque, Inc.), the plates wereincubated with culture supernatant followed by 1:2000 diluted
peroxidase-conjugated anti-mouse immunoglobulins (Agilent Technol­ogies, Inc., Santa Clara, CA). The enzymatic reaction was produced witha 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). The opticaldensity was measured at 655 nm using an iMark microplate reader (BioRad Laboratories, Inc., Berkeley, CA).

2. Materials and methods
2.1. Cell lines

2.5. Antibodies

The MIA PaCa-2 was obtained from the Cell Resource Center for the
Biomedical Research Institute of Development, Aging and Cancer
Tohoku University (Miyagi, Japan). P3U1 (mouse myeloma cell line)
and LN229 (glioblastoma cell line) were obtained from the American
Type Culture Collection (Manassas, VA). In our previous study (17), theLN229/PODXL and LN229/ectodomain-PODXL were produced. LN229,
LN229/PODXL, LN229/ectodomain-PODXL, and MIA PaCa-2 were
cultured in DMEM medium (Nacalai Tesque, Inc., Kyoto, Japan) andP3U1 was cultured in RPMI1640 medium (Nacalai Tesque, Inc.). The
media were supplemented with 10% heat-inactivated fetal bovine serum(FBS; Thermo Fisher Scientific Inc., Waltham, MA), 100 units/ml of
penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin
B (Nacalai Tesque, Inc.). The Vascular endothelial cells-1 and Vascularendothelial cells-2 were purchased from Cambrex Corp. (Walkersville,MD), and were cultured in endothelial cell medium EGM-2MV, supple­
mented with 5% FBS (Cambrex Corp.). All cells were cultured at 37 ◦ C ina humidified atmosphere containing 5% CO2 and 95% air.

PcMab-47, a mouse anti-PODXL mAb (IgG1, kappa), was developed
as previously described [17]. The mouse IgG was purchased from
Sigma-Aldrich Corp. (St. Louis, MO). To generate 60-mG2a, appropriate
VH cDNA of PcMab-60 and CH of mouse IgG2a were subcloned into
pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan), and VL and CL cDNAs of PcMab-60 were subcloned into
pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation). To
generate 60-mG2a, antibody expression vectors were transfected into
ExpiCHO-S cells using the ExpiCHO Expression System (Thermo Fisher
Scientific). To generate 60-mG2a-f, antibody expression vectors werealso transfected into BINDS-09 (FUT8-knocked out ExpiCHO-S cells)
using the ExpiCHO Expression System [23]. PcMab-60, 60-mG2a, and60-mG2a-f were purified using Protein G-Sepharose (GE Healthcare
Bio-Sciences, Pittsburgh, PA).
2.6. Flow cytometry

2.2. Animals

Cell lines were harvested via a brief exposure to 0.25% trypsin/1 mM
ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). Afterwashing with 0.1% BSA in PBS, the cells were treated with 10 μg/mL ofprimary mAbs for 30 min at 4 ◦ C, followed by treatment with Alexa
Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technol­ogy, Danvers, MA). Fluorescence data were collected using an EC800
Cell Analyzer (Sony Corp., Tokyo, Japan).

All animal experiments were performed in accordance with relevant
guidelines and regulations to minimize animal suffering and distress in
the laboratory. The Animal Care and Use Committee of Tohoku Uni­
versity approved all the animal experiments for hybridoma production.
Animal experiments for antitumor activity were approved by the Insti­
tutional Committee for Experiments of the Institute of Microbial
Chemistry. Mice were maintained in a pathogen-free environment (23 ±2 ◦ C, 55 ± 5% humidity) on 11 h light/13 h dark cycle with food andwater supplied ad libitum across the experimental period. Mice weremonitored for health and weight every 2 or 5 days during the 3-weekperiod of each experiment. The loss of original body weight to a point>25% and/or a maximum tumor size >3000 mm3 were identified as
humane endpoints for euthanasia. Mice were euthanized by cervicaldislocation; death was verified by respiratory and cardiac arrest.

2.7. Determination of binding affinity using flow cytometry
The MIA PaCa-2 cells (2 × 105) were resuspended in 100 μL ofserially diluted PcMab-60 and 60-mG2a-f (6 ng/mL to 100 μg/mL), fol­
lowed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG(1:200; Cell Signaling Technology). Fluorescence data were collectedusing a cell analyzer (EC800). The dissociation constant KD was obtainedby fitting the binding isotherms using the built-in one-site binding
models in GraphPad PRISM 6 (GraphPad Software, La Jolla, CA).

2.3. Hybridoma production
We immunized four-week-old female BALB/c mice (CLEA, Tokyo,
Japan) with the purified ectodomain of human PODXL (100 μg) together
with Imject Alum (Thermo Fisher Scientific Inc.) by intraperitoneal (i.p.)
injection. After several additional immunizations, a booster i.p. injectionof LN229/PODXL was given 2 days before the mice were euthanized bycervical dislocation, and spleen cells were harvested. The spleen cells
were fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indi­
anapolis, IN). Hybridomas were grown in RPMI 1640 medium including
L-glutamine with hypoxanthine, aminopterin, and thymidine selection
medium supplement (Thermo Fisher Scientific Inc.). Culture superna­tants were screened using enzyme-linked immunosorbent assay (ELISA)
for binding to the purified ectodomain of PODXL.

2.8. Antitumor activity of anti-PODXL antibodies
Five-week-old female BALB/c nude mice were purchased fromCharles River and used in experiments at 7 weeks of age. The cells (0.3mL of 1.33 × 108/mL in DMEM) were mixed with 0.5 mL of BD Matrigel
Matrix Growth Factor Reduced (BD Biosciences, San Jose, CA). A 100-μL
suspension (containing 5 × 106 cells) was injected subcutaneously into
the right flanks of nude mice. After 1 day, 100 μg of 60-mG2a-f or mouse
IgG in 100 μL PBS were injected into the peritoneal cavity of eachmouse. Additional antibodies were injected at days 8 and 15. The mice
were euthanized 22 days after cell implantation. All data were expressedas the mean ± SEM. Statistical analysis was performed using the TukeyKramer test. P < 0.05 was considered statistically significant.
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Biochemistry and Biophysics Reports 24 (2020) 100826

Fig. 1. Flow cytometry against cancer cells and normal cells using anti-PODXL mAbs (PcMab-47 and PcMab-60). LN229 (A), LN229/PODXL (B), MIA PaCa-2(C), VEC-1 (D), and VEC-2 (E) cells were treated with PcMab-47 (10 μg/mL), PcMab-60 (10 μg/mL), or control, followed by secondary antibodies. Vascularendothelial cells, VEC.

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Biochemistry and Biophysics Reports 24 (2020) 100826

Fig. 3. Determination of binding affinity of anti-PODXL antibodies(PcMab-60 and 60-mG2a-f) using flow cytometry. MIA PaCa-2 cells weresuspended in 100 μL of serially diluted antibodies (6 ng/mL to 100 μg/mL), andsecondary antibodies were then added. Fluorescence data were collected using
a cell analyzer. GeoMean, the geometric mean of fluorescence intensity.

Fig. 1E (left). In contrast, PcMab-60 did not react with VEC-1 (Fig. 1D,right) and VEC-2 (Fig. 1E, right), indicating that PcMab-60 iscancer-specific.

Fig. 2. Flow cytometry against MIA PaCa-2 cells using anti-PODXL mAbs
(PcMab-60, 60-mG2a, and 60-mG2a-f). MIA PaCa-2 cells were treated with
PcMab-60 (10 μg/mL), 60-mG2a (10 μg/mL), 60-mG2a-f (10 μg/mL), or control,
followed by secondary antibodies.

3.2. Production of the mouse IgG2a-type antibody PcMab-60

3. Results

Because the mouse IgG2a possesses high ADCC and complementdependent cytotoxicity (CDC) activities [24], we next developed
IgG2a-type of PcMab-60, which was named as 60-mG2a. To generate
60-mG2a, appropriate VH cDNA of PcMab-60 and CH of mouse IgG2awere subcloned into pCAG-Ble vector, and VL and CL cDNAs of PcMab-60
were subcloned into pCAG-Neo vector. Both vectors were transfected
into ExpiCHO-S cells, and 60-mG2a was purified from the supernatant.
We further produced a core-fucose-deficient type of 60-mG2a, which wasnamed as 60-mG2a-f using the BINDS-09 cell line (FUT8-knockout
Expi–CHO–S cell line). Next, the reactivity of 60-mG2a and 60-mG2a-f
was confirmed by flow cytometry using MIA PaCa-2 cells. As shown in
Fig. 2, PcMab-60, 60-mG2a, and 60-mG2a-f reacted strongly with MIA
PaCa-2 cells.

3.1. Development of anti-PODXL mAbs
First, we immunized mice with recombinant PODXL, which was
purified from the culture supernatant of LN229/ectodomain-PODXL. A
booster i.p. injection of LN229/PODXL was administered. The culture
supernatants were screened using ELISA for binding to purified PODXL.
As a second screening, we performed flow cytometry for reaction with
LN229 and LN229/PODXL, and a stronger reaction against LN229/
PODXL than against LN229 was necessary. As a positive control, we used
PcMab-47 [17], which strongly reacted with endogenous PODXL of
LN229 glioblastoma cell line (Fig. 1A, left). The reaction of PcMab-47 to
LN229/PODXL was slightly higher than that to LN229 (Fig. 1B, left). In
this study, we developed a novel anti-PODXL mAb, PcMab-60 (IgM,
kappa), which did not react with LN229 (Fig. 1A, right), but weakly
reacted with LN229/PODXL (Fig. 1B, right). In contrast, PcMab-60
showed a higher reaction with MIA PaCa-2 pancreatic cancer cell line
(Fig. 1C, right), although PcMab-47 reaction to MIA PaCa-2 is weaker
than that of LN229 (Fig. 1C, left), indicating that PcMab-60 might bespecific to pancreatic cancers. Because PODXL is highly expressed in
vascular endothelial cells, PcMab-47 stained PODXL of two vascularendothelial cells (VEC-1 and VEC-2) as shown in Fig. 1D (left) and3.3. The binding affinity of anti-PODXL mAbs
We performed a kinetic analysis of the interactions of PcMab-60 and
60-mG2a-f with MIA PaCa-2 cells using flow cytometry. As shown in
Fig. 3, the dissociation constant (KD) for PcMab-60 against MIA PaCa-2
was 4.9 × 10-8 M. In contrast, the KD for 60-mG2a-f was 9.1 × 10− 9 M.
The binding affinity of 60-mG2a-f against MIA PaCa-2 was 5.4-foldhigher than that of PcMab-60. These results are compatible with ourprevious observations that all the chimeric antibodies, including
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Biochemistry and Biophysics Reports 24 (2020) 100826

Fig. 5. Body weights of mice with MIA PaCa-2 xenografts. MIA PaCa-2 cells
were injected subcutaneously into female nude mice into the 60-mG2a-f group(n = 8) and the control IgG group (n = 8). The indicated antibodies (100 μg/
day; 5 mg/kg) were administered intraperitoneally 1, 8, and 15 days aftercancer cell inoculation. Body weights of mice with MIA PaCa-2 xenografts were
measured at the indicated time points. The values are means ± SEM. n.s., notsignificant by Tukey-Kramer’s test.

4. Discussion
In our previous study, we developed the original technology for the
production of cancer-specific monoclonal antibodies (CasMabs) against
membrane proteins [25–27]. We have successfully produced
anti-podoplanin CasMabs, such as LpMab-2 [25] and LpMab-23 [28]that specifically recognize cancer-type podoplanin in tumor tissues andnot in normal-type podoplanin, which is expressed in lymphatic vessels.
Moreover, the CasMab technology is useful for generating
anti-glycopeptide mAbs (GpMabs). We have produced the following
anti-podoplanin GpMabs: LpMab-3, LpMab-9, LpMab-12, LpMab-19,
and LpMab-21 [29–31]. The other original technology for the produc­tion of sensitive and specific mAbs is the Cell-Based Immunization andScreening (CBIS) method [32]. We have developed several
anti-podoplanin mAbs against pig [33], Tasmanian devil [34], alpaca[35], tiger [36], whale [37], goat [38], horse [39], and bear [40] PDPNs
using the CBIS method.
In this study, we employed the CasMab technology, and successfully
produced an anti-PODXL CasMab, PcMab-60, which highly reacts withMIA PaCa-2 pancreatic cell line, but did not react with PODXLexpressing vascular endothelial cells (Fig. 1). Although PcMab-60 was
determined to be IgM, which usually shows low binding affinity, it
showed moderate affinity (KD: 4.9 × 10-8 M) against MIA PaCa-2 cells(Fig. 3). Furthermore, 60-mG2a-f showed a higher binding affinity (KD:
9.1 × 10-9 M) compared with its original PcMab-60 (Fig. 3), indicating
derivatives of PcMab-60, such as humanized mAbs of PcMab-60, could
be useful for antibody-based therapy for pancreatic cancers. 60-mG2a-f
and 60-mG2a showed no difference in the binding affinity (data notshown). We have sometimes experienced that chimeric antibodies
possess much higher affinity or lower affinity when compared withoriginal mAbs [18,41–46]. The stability of antibodies might be different
among constant regions. We produced 60-mG2a-f, a non-fucosylated
version of 60-mG2a to augment its ADCC activities because
non-fucosylated antibodies are known to show higher ADCC activities[47,48]. As expected, 60-mG2a-f exhibited high antitumor activities in a
MIA PaCa-2 xenograft model (Fig. 4). In the future study, we need to
combine PcMab-60 with anti-cancer drugs or include them in novel
antitumor regimens, including T cells and viruses, to exert antitumor
activity against cancer cells.
High PODXL expression was significantly associated with worse OS
and was predictive of shorter OS in multiple cancers, especiallypancreatic cancers [49]. It was also revealed that high PODXL expres­sion predicted worse DSS and DFS. These results suggest that PODXL
could be a prognostic factor, and diagnostic tools targeting this protein

Fig. 4. Antitumor activity of 60-mG2a-f against MIA PaCa-2 xenograft. (A)Tumor volume of MIA PaCa-2 xenografts. MIA PaCa-2 cells were injectedsubcutaneously into female nude mice into the 60-mG2a-f group (n = 8) and thecontrol IgG group (n = 8). The indicated antibodies (100 μg/day; 5 mg/kg)
were administered intraperitoneally 1, 8, and 15 days after cancer cell inocu­
lation. The tumor volume was measured at the indicated time points. The
values are means ± SEM. (B) Tumor weight of MIA PaCa-2 xenografts (day 22).
The values are means ± SEM. An asterisk indicates statistical significance (*P <0.05, **P < 0.01, ***P < 0.005, Tukey-Kramer’s test). (C) Resected tumors of
MIA PaCa-2 xenografts (day 22).

chPcMab-47, 47-mG2a, and 47-mG2a-f, also possess a higher affinity for
PODXL than the original PcMab-47 [19].
3.4. Antitumor activity of 60-mG2a-f against MIA PaCa-2 xenografts
To investigate the antitumor effects of 60-mG2a-f on primary tumor
growth in vivo, the MIA PaCa-2 cells were subcutaneously implanted into
the flanks of nude mice. The 60-mG2a-f and the mouse IgG (control) were
injected three times (100 μg of the antibodies on days 1, 8, and 15 aftercell injections) into the peritoneal cavity of mice. Tumor formation wasobserved in all groups. The 60-mG2a-f significantly reduced tumor
development compared with mouse IgG on days 7, 10, 14, 17, 21, and 22(Fig. 4A). The tumor weights of mice in the 60-mG2a-f group weresignificantly lower than that in the IgG group on day 22 (Fig. 4B) The
resected tumors of MIA PaCa-2 xenografts are depicted in Fig. 4C. Body
weight was not significantly different between mouse IgG and 60-mG2a-fgroups (Fig. 5).

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Biochemistry and Biophysics Reports 24 (2020) 100826

are expected. Unfortunately, we herein could not analyze PODXL
expression in pancreatic cancer tissues because PcMab-60 was not usefulfor immunohistochemical analysis. In contrast, our previously estab­lished PcMab-47 was advantageous for immunohistochemical analysis[20]. The PODXL expression in pancreatic cancer tissues could beanalyzed using PcMab-47, and then PODXL-positive patients should betreated using cancer-specific humanized PcMab-60.

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Declaration of competing interest
The authors declare no conflicts of interest involving this article.
Acknowledgments
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:
17K07299 (M.K.K.) and 19K07705 (Y.K.).
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