cancers
ArticleA Humanized and Defucosylated Antibody against Podoplanin(humLpMab-23-f) Exerts Antitumor Activities in Human Lung
Cancer and Glioblastoma Xenograft Models
Hiroyuki Suzuki 1,2, *,† , Tomokazu Ohishi 3,4,† , Mika K. Kaneko 1,2
1

2

3

4

*
†

Citation: Suzuki, H.; Ohishi, T.;
Kaneko, M.K.; Kato, Y. A Humanized

and Yukinari Kato 1,2, *Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Miyagi, Japan; k.mika@med.tohoku.ac.jp
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
18-24 Miyamoto, Numazu-shi 410-0301, Shizuoka, Japan; ohishit@bikaken.or.jp
Institute of Microbial Chemistry (BIKAKEN), Laboratory of Oncology, Microbial Chemistry Research
Foundation, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-22-717-8207 (H.S. & Y.K.)
These authors contributed equally to this work.

Simple Summary: Podoplanin (PDPN), also known as T1α/Aggrus/gp36, is a type I transmembranesialo-glycoprotein that plays essential roles in cancer progression and metastasis. PDPN-expressing
cancers show aggressive phenotypes, including increased stemness, invasiveness, and epithelial-tomesenchymal transition, which lead to malignant progression. Furthermore, PDPN-positive cancerassociated fibroblasts mediate an immunosuppressive tumor microenvironment, which reduces
antitumor immunity. Therefore, monoclonal antibodies (mAbs) against PDPN have been evaluatedin preclinical models. In this study, we developed a humanized and defucosylated mAb against
PDPN (humLpMab-23-f) and evaluated its antibody-dependent cellular cytotoxicity (ADCC) andantitumor effect in xenograft models of human tumor cells. humLpMab-23-f exerted antitumor
activity against PDPN-overexpressed CHO-K1, endogenous PDPN-positive PC-10 (human lungsquamous cell carcinoma), and LN319 (human glioblastoma) xenograft-inoculated mice.

and Defucosylated Antibody against
Podoplanin (humLpMab-23-f) Exerts
Antitumor Activities in Human Lung
Cancer and Glioblastoma Xenograft
Models. Cancers 2023, 15, 5080.
https://doi.org/10.3390/
cancers15205080
Academic Editor: Gabriella D’Orazi
Received: 24 August 2023
Revised: 20 September 2023

Abstract: A cancer-specific anti-PDPN mAb, LpMab-23 (mouse IgG1 , kappa), was established in ourprevious study. We herein produced a humanized IgG1 version (humLpMab-23) and defucosylated
form (humLpMab-23-f) of an anti-PDPN mAb to increase ADCC activity. humLpMab-23 recognized
PDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/PDPN), PDPN-positive PC-10 (human lung squamous cell carcinoma), and LN319 (human glioblastoma) cells via flow cytometry. We
then demonstrated that humLpMab-23-f induced ADCC and complement-dependent cytotoxicity
against CHO/PDPN, PC-10, and LN319 cells in vitro and exerted high antitumor activity in mouse
xenograft models, indicating that humLpMab-23-f could be useful as an antibody therapy against
PDPN-positive lung squamous cell carcinomas and glioblastomas.

Accepted: 18 October 2023
Published: 20 October 2023

Keywords: podoplanin (PDPN); lung squamous cell carcinoma; glioblastoma; monoclonal antibody;
antitumor activity; mouse xenograft model; antibody-dependent cellular cytotoxicity; complementdependent cytotoxicity

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Podoplanin (PDPN), also known as T1α/gp36/Aggrus, is a type I transmembranesialo-glycoprotein that possesses an extracellular domain, transmembrane domain, andshort cytoplasmic tail [1]. The PDPN extracellular domain has tandem platelet aggregationstimulating domains, such as PLAG1, PLAG2, and PLAG3, which are associated with

Cancers 2023, 15, 5080. https://doi.org/10.3390/cancers15205080

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tumor-induced platelet aggregation [1]. A few PLAG-like domains (PLDs) have also been
identified, one of which is called the PLAG4 domain [1]. In the extracellular domains of
PDPN, serine or threonine residues are modified with mucin O-glycans with galactose β1,3-linked to N-acetyl-galactosamine (GalNAc) [1]. O-glycosylated PLAG3 and PLAG4 havebeen reported to interact with C-type lectin-like receptor 2 (CLEC-2), which is important for
PDPN-induced platelet aggregation [2,3]. The PDPN intracellular domain can recruit theezrin, radixin, and moesin (ERM) complex, which regulates actin cytoskeleton reorganization and epithelial-to-mesenchymal transition (EMT) [4]. Furthermore, PDPN interacts withmatrix metalloproteinases [5] and hyaluronan receptor CD44 [6]. The complex is formed intumor invadopodia, which promotes hyaluronan binding and extracellular matrix (ECM)
degradation [7]. Furthermore, PDPN mediates the diverse pattern of tumor invasion,
including collective invasion in squamous cell carcinomas [8] and ameboid invasion inmelanoma [9].
Overexpression of PDPN has been reported in many tumors, including squamous cell
carcinomas in the head and neck, esophagus, malignant gliomas, and mesotheliomas, and isassociated with poor clinical outcomes [1]. Furthermore, PDPN is also expressed in cancerassociated fibroblasts (CAFs), a major component of the tumor microenvironment (TME).
CAFs are reported to enhance cancer cell survival and affect therapeutic outcomes. CAFs arealso involved in the formation of an immunosuppressive TME, which can reduce antitumor
immunity [10]. Elevated PDPN staining in CAFs is correlated with poor prognosis inlung [11–13], breast [14], and pancreatic [15] cancer patients. Therefore, PDPN has been
considered as a useful marker for diagnosis and an attractive therapeutic target for tumors.
Our group previously established several mAbs against human PDPN. Clone NZ-1
exhibits neutralizing activity for CLEC-2 binding and prevents platelet aggregation andhematogenous metastasis to the lung [1]. An NZ-1-derived rat-human chimeric antibody,
named NZ-8 (human IgG1 , kappa), possesses antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cellular cytotoxicity (CDC) activities and exerts antitumor effects against malignant mesotheliomas [16]. A mouse-human chimeric anti-PDPN
mAb, named chLpMab-7 (human IgG1 , kappa), which recognizes the PLD domain, did
not show neutralization activity for CLEC-2 binding but suppressed tumor growth andhematogenous metastasis to the lung, indicating that ADCC/CDC activities are essential
for targeting PDPN-positive tumors [17].
To select mAbs that exhibit cancer specificity, we have established a cancer-specificmonoclonal antibody (CasMab) method [18]. One CasMab, clone LpMab-2, recognizes theglycopeptide structure of PDPN from Thr55 to Leu64 [18]. In contrast, another CasMabagainst PDPN, clone LpMab-23, recognizes a peptide structure of PDPN from Gly54 to
Leu64 [19]. We successfully applied a PDPN-targeting CasMab to chimeric antigen receptor(CAR)-T therapy in mice preclinical studies of human glioblastoma [20]. Recently, we
further developed anti-HER2 CasMabs [21].
ADCC activity is mediated by natural killer (NK) cells through the interaction of
FcγRIIIa with the Fc region of mAbs. The interaction between Fc and FcγRIIIa on effector
cells is facilitated by a core fucose deficiency in the N-glycan in the Fc region [22]. Therefore,fucosyltransferase 8 (Fut8)-deficient Chinese hamster ovary (CHO) cells are the ideal hosts
to produce completely defucosylated recombinant mAbs [23]. This technique was applied
to the development of mogamulizumab (Poteligeo® ), a defucosylated humanized mAbtargeting CCR4 [24]. We previously produced mouse-human chimeric and defucosylated
forms of anti-PDPN mAbs from LpMab-23 [19], which showed an antitumor effect in amouse xenograft model.
In this study, we produced a humanized and defucosylated anti-PDPN mAb (humLpMab23-f) and evaluated its ability to induce ADCC and CDC or antitumor efficacy against
PDPN-positive tumor cells.

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2. Materials and Methods
2.1. Cell Lines
CHO-K1 cells were obtained from the American Type Culture Collection (ATCC,
Manassas, VA, USA). CHO/PDPN cells were established as previously described [18].
Human lung squamous cell carcinoma PC-10 cells were purchased from Immuno-Biological
Laboratories Co., Ltd. (Gunma, Japan). CHO-K1, CHO/PDPN, and PC-10 cells were
cultured in Roswell Park Memorial Institute (RPMI)-1640 medium [Nacalai Tesque, Inc.
(Nacalai), Kyoto, Japan]. Human glioblastoma LN319 cells were purchased from Addexbio
Technologies (San Diego, CA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium(DMEM) (Nacalai). Both media were supplemented with 10% heat-inactivated fetal bovine
serum [FBS; Thermo Fisher Scientific, Inc. (Thermo), Waltham, MA, USA], 100 units/mL
penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (Nacalai). ExpiCHO-S
and Fut8-deficient ExpiCHO-S (BINDS-09) cells were cultured following the manufacturer’s
instructions. All cell lines were cultured at 37 ◦ C in a humidified atmosphere with 5% CO2
and 95% air.
2.2. Recombinant mAb Production
Normal human IgG was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA).
The complementarity determining region (CDR) of LpMab-23 VH , frame sequences of VH inhuman Ig, and CH of human IgG1 were cloned into the pCAG-Neo vector [FUJIFILM Wako
Pure Chemical Corporation (Wako), Osaka, Japan] to generate a humanized anti-human
PDPN mAb (humLpMab-23). The CDR of LpMab-23 VL , frame sequences of VL in humanIg, and CL of human kappa chain were cloned into the pCAG-Ble vector (Wako). We
transfected the antibody expression vectors of humLpMab-23 into ExpiCHO-S or BINDS-09
cells using the ExpiCHO-S Expression System (Thermo Fisher Scientific, Inc.). We named
these mAbs humLpMab-23 and humLpMab-23-f, respectively. We purified humLpMab-23
and humLpMab-23-f using Ab-Capcher (ProteNova Co., Ltd., Kagawa, Japan).
2.3. Animal Experiments for Mice Xenograft Model
To examine the antitumor effect of humLpMab-23-f, animal experiments were approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (approval no. 2020-035). During the experimental period, we monitored mice maintained in a pathogen-free environment on 11 h light/13 h dark cycle with food and watersupplied ad libitum. Mice were monitored for health and weight every one or five days. We
identified body weight loss exceeding 25% and maximum tumor size exceeding 3000 mm3
as humane endpoints, and terminated the experiments.
2.4. Flow Cytometry
CHO-K1, CHO/PDPN, PC-10, and LN319 cells were collected using 0.25% trypsin and
1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai). The cells (1 × 105 cells/sample)
were treated with humLpMab-23 or blocking buffer (control) (0.1% BSA in PBS) for 30 min
at 4 ◦ C. Next, the cells were treated with fluorescein isothiocyanate (FITC)-conjugatedanti-human IgG (1:2000; Sigma-Aldrich Corp., St. Louis, MO, USA) for 30 min at 4 ◦ C.
The EC 800 Cell Analyzer (Sony Corp., Tokyo, Japan) was used to collect the fluorescencedata, which were analyzed using FlowJo software [BD Biosciences (BD), Franklin Lakes,
NJ, USA].
2.5. Determination of the Binding Affinity by Flow Cytometry
After being suspended in 100 µL of serially diluted humLpMab-23, the cells werethen incubated with FITC-conjugated anti-human IgG (1:200). The EC800 Cell Analyzer
was then used to gather fluorescence data. The binding isotherms were fitted into thebuilt-in, one-site binding model using GraphPad PRISM 8 software (GraphPad Software,Inc., La Jolla, CA, USA) to calculate the dissociation constant (KD ).

Cancers 2023, 15, 5080

4 of 132.6. ADCC
We purchased human NK cells from Takara Bio, Inc. (Shiga, Japan). We labeled
target cells (CHO/PDPN, PC-10, and LN319) using 10 µg/mL Calcein AM (Thermo
Fisher Scientific, Inc.) and resuspended them. Target cells were plated in 96-well plates(1 × 104 cells/well) and mixed with the human NK cells and 100 µg/mL of control humanIgG or humLpMab-23-f. The calcein released to the supernatant from each well was measured after a 4.5 h incubation. Fluorescence intensity was determined using a microplate
reader (Power Scan HT; BioTek Instruments, Winooski, VT, USA) with excitation andemission wave lengths of 485 and 538 nm, respectively. After lysing all cells with a buffer
containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4), and 10 mM EDTA, cytotoxicity (%lysis) was calculated as % lysis = (E − S)/(M − S) × 100, where E is the fluorescence of the
combined target and effector cells, S is the spontaneous fluorescence of target cells only,
and M is the maximum fluorescence measured.
2.7. CDC
The calcein AM-labeled target cells (CHO/PDPN, PC-10, and LN319) were plated
and mixed with rabbit complement (final dilution 1:10, Low-Tox-M Rabbit Complement;
Cedarlane Laboratories, Hornby, ON, Canada) and 100 µg/mL of control human IgG or
humLpMab-23-f. Following incubation for 4.5 h at 37 ◦ C, the calcein released into themedium was measured, as described above.
2.8. Antitumor Activity of humLpMab-23-f in Xenografts of CHO-K1, CHO/PDPN, PC-10, and
LN319 Cells
First, each cell was suspended in 0.3 mL of 1.33 × 108 cells/mL using DMEM andmixed with 0.5 mL of BD Matrigel Matrix Growth Factor Reduced (BD Biosciences, San Jose,
CA, USA). Then, BALB/c nude mice (Jackson Laboratory Japan, Kanagawa, Japan) were
injected subcutaneously in the left flank with 100 µL of the suspension (5 × 106 cells). After
day six, we injected the mice with 100 µg of humLpMab-23-f (n = 8) and control human
IgG (n = 8) in 100 µL of PBS through intraperitoneal injection (i.p.). Additional antibodies
were injected on days 14 and 21.
Human NK cells (8.0 × 105 cells) were injected around the tumors on days 6, 14, and 21.
The tumor diameter was measured on days 6, 11, 14, 18, 21, and 25 after inoculation withcells. The tumor volume was calculated using the following formula: volume = W2 × L/2,
where W is the short diameter and L is the long diameter. All mice were euthanized bycervical dislocation 25 days after cell inoculation.
2.9. Statistical Analyses
All data are represented as the mean ± standard error of the mean (SEM). Welch’s
t-test was used for the statistical analyses of ADCC, CDC, and tumor weight. ANOVA
with Sidak’s post hoc test was used for tumor volume and mouse weight. p < 0.05 wasconsidered to indicate a statistically significant difference.
3. Results
3.1. Production of Humanized Anti-PDPN mAb, humLpMab-23
We previously established an anti-PDPN mAb (LpMab-23; mouse IgG1 , kappa) byimmunization with the PDPN ectodomain produced by glioblastoma LN229 cells [19].
LpMab-23 was shown to be useful for flow cytometry [19]. In this study, we engineereda humanized LpMab-23 (humLpMab-23) by fusing the VH and VL CDRs of LpMab-23
with the CH and CL chains of human IgG1 , respectively (Figure 1A). humLpMab-23 wasdetected in CHO/PDPN cells, but not in parental CHO-K1 cells (Figure 1B). Furthermore,
humLpMab-23 reacted with PDPN-positive glioblastoma LN319 and lung squamous cell
carcinoma PC-10 cells (Figure 1C).

Cancers 2023, 15, 5080

humLpMab-23 reacted with PDPN-positive glioblastoma LN319 and lung squamous cell
carcinoma PC-10 cells (Figure 1C).
The KD for the interaction of humLpMab-23 with CHO/PDPN, LN319, and PC-10
cells was determined by flow cytometry. As shown in Figure 1D, the KD values for
humLpMab-23 with CHO/PDPN, LN319, and PC-10 cells were 4.7 × 10−9 M, 4.9 × 10−9 M,
5 of 13and 5.4 × 10−9 M, respectively. These results suggested that humLpMab-23-f demonstrated
a high aﬃnity for PDPN-positive cells.

Figureusing
humLpMab-23.
(A) (A)A humanized
IgG1IgGmAb,mAb,
humLpMab-23,
was wasFigure1.1.Flow
Flowcytometry
cytometryusing
humLpMab-23.
A humanizedhumLpMab-23,
1
generated from LpMab-23-f (mouse IgG1). The core fucose-deficient form (humLpMab-23-f) was
generated from LpMab-23-f (mouse IgG1 ). The core fucose-deficient form (humLpMab-23-f) was
produced using Fut8-knockout ExpiCHO-S cells. (B) CHO/PDPN and CHO-K1 cells were treatedproducedusing Fut8-knockout
ExpiCHO-S(B) byCHO/PDPN
CHO-K1 cellswith humLpMab-23(1 µg/mL) or buﬀer
control, cells.
followedanti-humanand
IgG conjugatedwithwere
FITC.treatedwithhumLpMab-23(1µg/mL)
orbuffercontrol,followedbyanti-human
IgG
conjugated(C) LN319 and PC-10 cells were treated with humLpMab-23 (1 µg/mL) or buﬀer control, followed with
FITC.
(C)Fluor
LN319
and PC-10 cells
were treated
withDetermination
humLpMab-23
buffer ofcontrol,byAlexa
488-conjugatedanti-human
IgG. (D)of (1theµg/mL)bindingoraﬃnity

followed by Alexa Fluor 488-conjugated anti-human IgG. (D) Determination of the binding affinityof humLpMab-23 using flow cytometry. CHO/PDPN, LN319, and PC-10 cells were suspended in
humLpMab-23 at indicated concentrations, followed by treatment with anti-human IgG conjugatedwith FITC. The EC800 Cell Analyzer was used to analyze fluorescence data. GraphPad Prism 8 wasused to determine the dissociation constant (KD ).

The KD for the interaction of humLpMab-23 with CHO/PDPN, LN319, and PC-10 cells
was determined by flow cytometry. As shown in Figure 1D, the KD values for humLpMab23 with CHO/PDPN, LN319, and PC-10 cells were 4.7 × 10−9 M, 4.9 × 10−9 M, and
5.4 × 10−9 M, respectively. These results suggested that humLpMab-23-f demonstrated ahigh affinity for PDPN-positive cells.
3.2. ADCC and CDC by Humlpmab-23-f against CHO/PDPN Cells
Next, we developed the core fucose-deficient version of humLpMab-23, named
humLpMab-23-f, using BINDS-09 cells (Figure 1A). We then examined if humLpMab-23-f
showed ADCC activity against CHO/PDPN cells. As shown in Figure 2A, humLpMab-23-f
induced ADCC against CHO/PDPN cells (51.6% cytotoxicity) more effectively than thecontrol human IgG (13.6% cytotoxicity; p < 0.05). No difference was observed in ADCC

Cancers 2023, 15, 5080

the control human IgG (13.6% cytotoxicity; p < 0.05). No diﬀerence was observed in ADCC
against CHO-K1 cells between the humLpMab-23-f and control human IgG-treatedgroups (Figure 2B).
Furthermore, we investigated CDC by humLpMab-23-f against CHO/PDPN cells. As
shown in Figure 2C, humLpMab-23-f induced greater CDC against CHO/PDPN cells(69.2% cytotoxicity) compared with that induced by control human IgG (15.0%6 of 13
cytotoxicity; p < 0.05). No diﬀerence was observed in CDC against CHO-K1 cells between
the humLpMab-23-f and control human IgG-treated groups (Figure 2D).
These
resultscells
demonstrated
that
humLpMab-23-fandexhibited
ADCC
and CDCagainstCHO-K1
between the
humLpMab-23-f
controlpotent
human
IgG-treatedgroupsactivities
against
CHO/PDPN
cells.
(Figure 2B).

Figure 2.
mediated
by by
humLpMab-23-f
in CHO-K1
and CHO/PDPN
Figure2. TheTheADCC
ADCCand
andCDC
CDCactivities
activities
mediated
humLpMab-23-f
in CHO-K1
and CHO/PDPN
cells. (A,B) The ADCC induced by humLpMab-23-f or control human IgG against CHO/PDPN (A)cells. (A,B) The ADCC induced by humLpMab-23-f or control human IgG against CHO/PDPN
and CHO-K1 (B) cells. (C,D) The CDC induced by humLpMab-23-f or control human IgG against(A)
and CHO-K1(B) CHO-K1
cells. (C,D)
CDC
inducedby humLpMab-23-f
controlhuman
IgG against(D)Thecells.
Valuesare shownas the mean ± or
SEM.
Asterisks
indicate
CHO/PDPN(C) and
CHO/PDPN(C) and(*CHO-K1(D) cells.
Valuesas the mean ± SEM. Asterisks indicate
statistical significancep < 0.05; Welch’s
t-test).
n.s.,arenotshown
significant.
statistical significance (* p < 0.05; Welch’s t-test). n.s., not significant.

3.3. Antitumor Eﬀects of Humlpmab‐23‐f against CHO/PDPN Xenografts

Furthermore, wehumLpMab-23-f
investigated CDC
humLpMab-23-f
CHO/PDPN
Intraperitoneally,
andby
controlhuman IgG against
were administeredon cells.
As
shown
in
Figure2C,
humLpMab-23-f
inducedgreaterCDC
against
CHO/PDPN
days 6, 14, and 21, following inoculation with CHO/PDPN cells in BALB/c nude mice. cells(69.2% cytotoxicity) compared with that induced by control human IgG (15.0% cytotoxicity; p < 0.05). No difference was observed in CDC against CHO-K1 cells between the
humLpMab-23-f and control human IgG-treated groups (Figure 2D).
These results demonstrated that humLpMab-23-f exhibited potent ADCC and CDC
activities against CHO/PDPN cells.
3.3. Antitumor Effects of Humlpmab-23-f against CHO/PDPN Xenografts
Intraperitoneally, humLpMab-23-f and control human IgG were administered ondays 6, 14, and 21, following inoculation with CHO/PDPN cells in BALB/c nude mice.
Furthermore, human NK cells were injected around the tumors on the same days as themAb injection. The tumor volume was measured on days 6, 11, 14, 18, 21, and 25 after
inoculation with CHO/PDPN cells. humLpMab-23-f administration resulted in a significant
reduction in tumor volume on days 18 (p < 0.01), 21 (p < 0.01), and 25 (p < 0.01) comparedwith that induced by the human IgG (Figure 3A). humLpMab-23-f treatment resulted inabout 73% reduction in volume compared with that induced by the control human IgG
on day 25 post-injection. When treated with humLpMab-23-f, the weight of CHO/PDPN
tumors was significantly lower compared with those treated with control human IgG (90%reduction; p < 0.01; Figure 3C). Figure 3E demonstrates the CHO/PDPN tumors resectedfrom mice on day 25. Body weight was not affected in CHO/PDPN tumor-bearing micetreated with humLpMab-23-f and control human IgG (Figure 3G).

Cancers 2023, 15, 5080

compared with that induced by the human IgG (Figure 3A). humLpMab-23-f treatment
resulted in about 73% reduction in volume compared with that induced by the control
human IgG on day 25 post-injection. When treated with humLpMab-23-f, the weight of
CHO/PDPN tumors was significantly lower compared with those treated with control
human IgG (90% reduction; p < 0.01; Figure 3C). Figure 3E demonstrates the CHO/PDPN7 of 13
tumors resected from mice on day 25. Body weight was not aﬀected in CHO/PDPN tumorbearing mice treated with humLpMab-23-f and control human IgG (Figure 3G).

Figure3.3. The
The antitumor
antitumor eﬀect
CHO-K1andandCHO/PDPN
xenografts.
Figureeffectof
ofhumLpMab-23-f
humLpMab-23-fagainst
againstCHO-K1
CHO/PDPN
xenografts.
(A,B) CHO/PDPN (A) and CHO-K1 (B) cells were subcutaneously inoculated into BALB/c nude(A,B) CHO/PDPN (A) and CHO-K1 (B) cells were subcutaneously inoculated into BALB/c nude mice(day 0). On day 6, 100 µg of humLpMab-23-f or control human IgG was administered intraperitoneally.
Additional antibodies were administered on days 14 and 21. Human NK cells were injected aroundthe tumors on the same days as mAb administration. Tumor volume was measured on days 6, 11, 14,
18, 21, and 25. Values are presented as the mean ± SEM. ** p < 0.01 (ANOVA and Sidak’s multiple
comparisons test). (C,D) Weight of CHO/PDPN (C) and CHO-K1 (D) xenograft tumors on day 25.
Values are represented as the mean ± SEM. ** p < 0.01 (Welch’s t-test). (E,F) The CHO/PDPN (E) andCHO-K1 (F) xenograft tumors on day 25 (scale bar, 1 cm). (G,H) Body weight of CHO/PDPN (G) andCHO-K1 (H) xenograft-bearing mice treated with control human IgG and humLpMab-23-f. n.s.,
not significant.

In the CHO-K1-inoculated mice, humLpMab-23-f and control human IgG were injected
intraperitoneally into mice on days 6, 14, and 21 after inoculation. Furthermore, human NK
cells were injected around the tumors on the same days as the mAb injection. There was nodifference in CHO-K1 xenograft volume (Figure 3B) and weight (Figure 3D) between theCancers 2023, 15, 5080

8 of 13

humLpMab-23-f and control human IgG-treated groups. Figure 3F demonstrates the CHOK1 tumors that were resected from mice on day 25. Body weight loss was not observed(Figure 3H). Supplementary Figure S1 presents the body appearance of CHO/PDPN and
CHO-K1 xenograft-inoculated mice treated with human IgG and humLpMab-23-f on
day 25.
3.4. ADCC and CDC by Humlpmab-23-f against LN319 and PC-10 Cells
We next investigated ADCC by humLpMab-23-f against endogenous PDPN-positive
LN319 and PC-10 cells. As shown in Figure 4A, humLpMab-23-f showed more potent
ADCC against LN319 cells (47.7% cytotoxicity) than that induced by control human IgG(15.8% cytotoxicity; p < 0.01). Furthermore, humLpMab-23-f showed more potent ADCC
Cancers 2023, 15, x FOR PEER REVIEW
9 of 14
against PC-10 cells (46.7% cytotoxicity) than that induced by control human IgG(14.6%
cytotoxicity; p < 0.01) (Figure 4B).

Figure 4.
4. Themediated
by by
humLpMab-23-f
in PDPN-positive
LN319
and andFigureTheADCC
ADCCand
andCDC
CDCactivities
activities
mediated
humLpMab-23-f
in PDPN-positive
LN319
against
LN319(A) and
PC-10 cells.
cells. (A,B)
or control
human
IgG IgG
PC-10(A,B)ADCC
ADCCinduced
inducedbybyhumLpMab-23-f
humLpMab-23-f
or control
humanagainst
LN319(A) and
PC-10 (B) cells. (C,D) CDC induced by humLpMab-23-f or control human IgG against LN319 (C)PC-10 (B) cells. (C,D) CDC induced by humLpMab-23-f or control human IgG against LN319 (C) andand PC-10 (D) cells. Values are shown as the mean ± SEM. Asterisks indicate statistical significancePC-10(D) **
cells.
Valuesare shownas the± SEM. Asterisks indicate statistical significancet-test). n.s.,
not meansignificant.
(* p < 0.05,
p < 0.01;
Welch’s(* p < 0.05, ** p < 0.01; Welch’s t-test).

3.5. Antitumor Eﬀects of Humlpmab‐23‐f in LN319 and PC‐10 Xenografts

For CDC by humLpMab-23-f against LN319 cells, humLpMab-23-f exhibited greater
In the LN319 and PC-10 xenograft tumor-bearing mice, humLpMab-23-f and control
CDC (34.7% cytotoxicity) compared with that induced by control human IgG (11.6% cytohuman IgG were intraperitoneally administered on days 6, 14, and 21. Furthermore,
toxicity;p <cells
0.05)(Figure4C).around
Moreover,humLpMab-23-f
exhibitedgreater
CDC against
administration.
human NK
were
injected
the tumors
on the same days
as mAb
PC-10
cells(53.4%
cytotoxicity)
comparedwiththat
inducedbycontrolhuman
IgG (15.0%
The tumor volume was measured on days 6, 11, 14, 18, 21, and 25 after inoculation.
cytotoxicity;
p
<
0.01)
(Figure4D).
humLpMab-23-f treatment significantly reduced the volume of LN319 xenografts on days
results
demonstrated
that humLpMab-23-f
exhibited
21 (pThese< 0.01)and 25(p < 0.01) comparedwith that inducedby the potent
controlADCC
humanandIgGCDC
activities
againstendogenous
PDPN-positive
LN319
and
PC-10
cells.
(Figure 5A). humLpMab-23-f also induced a significant reduction in PC-10 xenograftvolume on days 18 (p < 0.01), 21 (p < 0.01), and 25 (p < 0.01) compared with that induced

3.5.
Antitumor
Effects IgGof Humlpmab-23-f
in LN319 and PC-10
Xenografts
by thecontrol human(Figure 5B). humLpMab-23-f
treatment
resulted in 64% (LN319)
the(PC-10)
LN319decrease
and PC-10
xenografttumor-bearing
mice,
humLpMab-23-f
controland In69%
in tumorweightcompared withthat
induced by the andcontrolhumanintraperitoneallyadministeredondemonstrates
days 6, 14, andFurthermore,human
human IgG
IgGwereon day25 (Figure 5C,D).
Figure 5E,F
the21.LN319
and PC-10
tumorswere
resectedon daythe
25.tumors on the same days as mAb administration. The
NK
cellsthatwere
injectedaroundweightwas not on
observed
either
LN319
or PC-10
xenograft-bearing
mice
tumorBody
volumewasloss
measureddays 6,in11,
14, 18,21, and25 after
inoculation. humLpMabtreatedwith humLpMab-23-f
and control
humanofIgG(Figure5G,H). on
Supplementary23-ftreatment
significantly reduced
the volumeLN319
xenografts
days 21 (p < 0.01)
Figure S2 presents the body appearance of LN319 and PC-10 xenograft-inoculated micetreated with human IgG and humLpMab-23-f on day 25.

Cancers 2023, 15, 5080

9 of 13and 25 (p < 0.01) compared with that induced by the control human IgG (Figure 5A).
humLpMab-23-f also induced a significant reduction in PC-10 xenograft volume on days 18(p < 0.01), 21 (p < 0.01), and 25 (p < 0.01) compared with that induced by the control human
IgG (Figure 5B). humLpMab-23-f treatment resulted in 64% (LN319) and 69% (PC-10)
decrease in tumor weight compared with that induced by the control human IgG on day 25
Cancers 2023, 15, x FOR PEER REVIEW
10 of 14
(Figure 5C,D). Figure 5E,F demonstrates the LN319 and PC-10 tumorsthat were resectedon day 25.

Figure 5. Antitumor activity of humLpMab-23-f against LN319 and PC-10 xenografts. (A,B) LN319

Figure5. Antitumor activity of humLpMab-23-f against LN319 and PC-10 xenografts. (A,B) LN319(A) and PC-10 (B) cells were subcutaneously injected into BALB/c nude mice (day 0). On day 6, 100(A)
PC-10 (B) cells
were subcutaneously
injected into
BALB/cnude miceµg andof humLpMab-23-f
or control
human IgG was administered.
Additional
antibodies
were (day 0). On day 6,
Human
NK humancells wereIgG
injected
the tumors on
the same antibodies wereadministeredon days 14 and 21.or
100µg of humLpMab-23-f
control
wasaroundadministered.
Additionaldays as mAb administration. The tumor volume was measured on days 6, 11, 14, 18, 21, and 25.
administeredon daysand± 21.
cells were
injectedaroundthe tumors on the same
Values are presented
as the14
mean
SEM.Human** p < 0.01NK(ANOVAand Sidak’s
multiplecomparisons
Weightof LN319 (C) and PC-10(D) xenograft
tumorson measured
day 25. Values
representedtest). (C,D)
days
as mAbadministration.
The tumor
volumewasonaredays
6, 11, 14, 18, 21, and 25.
as the mean ± SEM. * p < 0.05; ** p < 0.01 (Welch’s t‐test). (E,F) The LN319 (E) and PC-10 (F) xenograft
Valuesarepresented
as
the
mean
±
SEM.
**
p
<
0.01
(ANOVAand
Sidak’smultiplecomparisons test).
tumors on day 25 (scale bar, 1 cm). (G,H) Body weight of LN319 (G) and PC-10 (H) xenograft(C,D)
Weightof LN319(C) andPC-10(D)
xenograft tumors
day 25. Values are represented as
bearing
mice treatedwith control
human
IgG andhumLpMab-23-f.
n.s., noton
significant.
the mean ± SEM. * p < 0.05; ** p < 0.01 (Welch’s t-test). (E,F) The LN319 (E) and PC-10 (F) xenograft
tumors on day 25 (scale bar, 1 cm). (G,H) Body weight of LN319 (G) and PC-10 (H) xenograft-bearing
mice treated with control human IgG and humLpMab-23-f. n.s., not significant.

Cancers 2023, 15, 5080

10 of 13

Body weight loss was not observed in either LN319 or PC-10 xenograft-bearing micetreated with humLpMab-23-f and control human IgG (Figure 5G,H). Supplementary Figure S2
presents the body appearance of LN319 and PC-10 xenograft-inoculated mice treated withhuman IgG and humLpMab-23-f on day 25.
4. Discussion
In this study, we showed that humLpMab-23-f exhibited potent ADCC and CDC
activities against CHO/PDPN, PC-10, and LN319 cells (Figures 2 and 4). Furthermore, administration of humLpMab-23-f in tumor-bearing mice completely suppressed the growthof CHO/PDPN xenografts (Figure 3A,C,E) and exerted potent antitumor effects in PC-10
and LN319 xenografts (Figure 5). These results indicated that humLpMab-23-f could be
useful as an antibody therapy for PDPN-positive human tumors.
We previously evaluated defucosylated mouse-human chimeric LpMab-23 (chLpMab23-f) [19]. Although chLpMab-23-f exhibited ADCC activity and exerted a moderate
antitumor effect against CHO/PDPN cells, chLpMab-23-f neither exhibited ADCC activity
against PC-10 cells nor CDC activity against PDPN-expressing tumor cells [19]. The resultsof the binding affinity experiments revealed that the KD of humLpMab-23 with PC-10
cells was 5.4 × 10−9 M (Figure 1D). In contrast, the KD of chLpMab-23 with PC-10 cells
was 1.4 × 10−7 M (Supplementary Figure S3). These results indicated that humLpMab-23
shows a superior binding affinity compared to chLpMab-23. The increased binding affinity
is thought to be a reason why humLpMab-23-f exerted more potent antitumor effects thanchLpMab-23.
PDPN is involved in tumor malignant progression through activation of tumor invasiveness, EMT, and stemness. Furthermore, PDPN induces platelet aggregation throughinteraction with CLEC-2 on platelets, which is associated with venous thromboembolism
and evasion of immune cells [25,26]. Therefore, several anti-PDPN mAbs have been evaluated in preclinical studies. Our group demonstrated that chLpMab-7 (a mouse-humanchimeric anti-PDPN mAb) and NZ-8 (a rat-human chimeric antibody derived from NZ-1)
exerted antitumor effects through their ADCC and CDC activities. Furthermore, NZ-1R700 conjugate [27] and NZ-16 labeled with actinium-225 (225 Ac) [28] have been developed
as near-infrared photoimmunotherapy and radioimmunotherapy agents against PDPN,
respectively. Both mAbs also exhibited antitumor effects against the malignant pleural mesothelioma xenograft model. In addition, neutralizing mAbs for PDPN-CLEC-2
interaction suppressed hematogenous pulmonary metastasis by inhibiting platelet aggregation [29]. LpMab-23 has a different epitope from the abovementioned anti-PDPN
mAbs [19] and showed comparable ADCC/CDC activities and antitumor effects in vivo
(Figures 2–5). In contrast, the reactivity of LpMab-23 to normal cells such as lymphatic
endothelial cells was low in immunohistochemical analysis [30]. It should be determined
whether humLpMab-23-f induces reduced cytotoxicity against normal PDPN-expressing
cells. We already confirmed that mouse-human chimeric LpMab-23 (20 mg/kg) [19] did
not show any toxicity against cynomolgus monkeys.
CAFs are important players in the TME. CAFs are involved in tumor progression
through the promotion of tumor proliferation, invasion, and chemoresistance, as well as
ECM remodeling and immunosuppression [31]. PDPN expression is also observed in CAFs.
Increased expression of PDPN in CAFs from lung [11–13], pancreas [15], and breast [14]tumors is associated with poor prognosis of patients. In PDPN-positive CAFs from lungtumors, the CAFs promote lung cancer cell resistance to inhibitors of EGFR [32]. Furthermore, increased expression of TGF-β was observed in PDPN-positive CAFs [13], which
are associated with an immunosuppressive TME [10]. In breast cancer, the S100A4/PDPN
expression ratio in CAFs is associated with disease outcomes across subtypes. High PDPN
levels in CAFs were significantly correlated with shorter recurrence-free survival and overall survival [33]. Moreover, PDPN-positive CAFs were associated with low IL-2 activity
and resistance to trastuzumab in HER2-positive breast cancer patients [34]. These findings
indicate that the character of CAFs is an important predictive marker of breast cancer

Cancers 2023, 15, 5080

11 of 13

progression and resistance to treatment. Therefore, targeting PDPN-positive CAFs using
anti-PDPN mAbs could be a strategy for tumor therapy. Since we have not examined thereactivity of humLpMab-23 to CAFs, further studies to characterize humLpMab-23-reactive
CAFs are needed.
PD-1 and CTLA-4 on effector T lymphocytes play essential functions in tumor immune response. PDPN was reported as a co-inhibitory receptor on both CD4+ and CD8+
T lymphocytes. In T lymphocyte-specific Pdpn-deleted mice, the growth of inoculated
B16F10 tumors was significantly delayed. The PDPN-deficient CD8+ tumor-infiltrating
lymphocytes exhibited increased tumor necrosis factor production, while the abundanceof exhausted T lymphocytes was decreased [35]. Moreover, deletion of Pdpn in T cells
resulted in the development of autoimmune encephalomyelitis with increased accumulation of effector CD4+ T lymphocytes [36]. These results suggest the involvement of PDPN
in tumor immune suppression. Although the ligand of PDPN involved in suppressing
T lymphocytes has not been identified, a PDPN-targeting mAb has potential as a novel
immune checkpoint inhibitor. Therefore, the combinatorial effects of anti-PDPN mAbs
with immune checkpoint inhibitors should be considered in preclinical studies. We haveestablished anti-PDPN mAbs against various species, including mice [1]. The anti-mouse
PDPN mAbs could be useful in revealing the importance of PDPN-positive lymphocytes incancer immunity and obtaining proof-of-concept for tumor therapy.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers15205080/s1, Figure S1. Body appearance in CHO/PDPN(A) and CHO-K1 (B) xenografts-inoculated mice treated with human IgG and humLpMab-23-f on
day 25; Figure S2. Body appearance in LN319 (A) and PC-10 (B) xenografts-inoculated mice treatedwith human IgG and humLpMab-23-f on day 25; Figure S3. Determination of the binding affinity of
chLpMab-23 using flow cytometry.
Author Contributions: H.S. and T.O. performed the experiments. M.K.K. and Y.K. designed theexperiments. H.S. and Y.K. analyzed the data. H.S. and Y.K. wrote the manuscript. All authors haveread and agreed to the published version of the manuscript.
Funding: This research was supported in part by Japan Agency for Medical Research and Development (AMED) under grant numbers JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.), 23bm1123027h0001(to Y.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.), and by the Japan Society for the
Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) under grant numbers
22K06995 (to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Institutional Review Board Statement: Animal experiments were approved by the Institutional
Committee for Experiments of the Institute of Microbial Chemistry (approval no. 2020-035).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article and Supplementary Materials.
Acknowledgments: The authors would like to thank Shun-ichi Ohba and Akiko Harakawa (Instituteof Microbial Chemistry [BIKAKEN], Numazu, the Microbial Chemistry Research Foundation) for
technical assistance with animal experiments.
Conflicts of Interest: The authors declare no conflict of interest.

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