Article

A Cancer-Speciﬁc Monoclonal Antibody against Podocalyxin
Exerted Antitumor Activities in Pancreatic Cancer Xenografts
Hiroyuki Suzuki 1,2,*, Tomokazu Ohishi 3,4, Tomohiro Tanaka 1,2, Mika K. Kaneko 1,2 and Yukinari Kato 1,2,*
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; tomohiro.tanaka.b5@tohoku.ac.jp (T.T.);mika.kaneko.d4@tohoku.ac.jp (M.K.K.)
2 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
3 Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
18-24 Miyamoto, Numazu-shi 410-0301, Japan; ohishit@bikaken.or.jp
4 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.)
1

Citation: Suzuki, H.; Ohishi, T.;
Tanaka, T.; Kaneko, M.K.; Kato, Y. A
Cancer-Speciﬁc Monoclonal
Antibody against Podocalyxin
Exerted Antitumor Activities in
Pancreatic Cancer Xenografts. Int. J.
Mol. Sci. 2024, 25, 161. https://doi.org/10.3390/ijms25010161
Academic Editor: Dorota Lubgan
Received: 27 November 2023

Abstract: Podocalyxin (PODXL) overexpression is associated with poor clinical outcomes in various
tumors. PODXL is involved in tumor malignant progression through the promotion of invasivenessand metastasis. Therefore, PODXL is considered a promising target of monoclonal antibody (mAb)based therapy. However, PODXL also plays an essential role in normal cells, such as vascular andlymphatic endothelial cells. Therefore, cancer speciﬁcity or selectivity is required to reduce adverse
eﬀects on normal cells. Here, we developed an anti-PODXL cancer-speciﬁc mAb (CasMab), PcMab6 (IgG1, kappa), by immunizing mice with a soluble PODXL ectodomain derived from a glioblastoma LN229 cell. PcMab-6 reacted with the PODXL-positive LN229 cells but not with PODXLknockout LN229 cells in ﬂow cytometry. Importantly, PcMab-6 recognized pancreatic ductal adenocarcinoma (PDAC) cell lines (MIA PaCa-2, Capan-2, and PK-45H) but did not react with normal
lymphatic endothelial cells (LECs). In contrast, one of the non-CasMabs, PcMab-47, showed highreactivity to both the PDAC cell lines and LECs. Next, we engineered PcMab-6 into a mouse IgG2atype (PcMab-6-mG2a) and a humanized IgG1-type (humPcMab-6) mAb and further produced thecore fucose-deﬁcient types (PcMab-6-mG2a-f and humPcMab-6-f, respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC). Both PcMab-6-mG2a-f and humPcMab-6-f exerted
ADCC and complement-dependent cellular cytotoxicity in the presence of eﬀector cells and complements, respectively. In the PDAC xenograft model, both PcMab-6-mG2a-f and humPcMab-6-f exhibited potent antitumor eﬀects. These results indicated that humPcMab-6-f could apply to antibody-based therapy against PODXL-expressing pancreatic cancers.
Keywords: podocalyxin; PODXL; cancer-speciﬁc monoclonal antibody; defucosylated antibody;
pancreatic cancer

Revised: 15 December 2023
Accepted: 19 December 2023
Published: 21 December 2023

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

1. Introduction
Podocalyxin (PODXL) is a CD34-related transmembrane sialomucin glycoprotein [1].
The 53-kDa PODXL core protein undergoes extensive N- and O-linked glycosylation,
leading to a mature protein with a molecular weight of 150,000–200,000 [2]. PODXL isnormally expressed by embryonic stem cells [3], early hematopoietic progenitors [4], adultvascular/lymphatic endothelial cells [5], and kidney podocytes [6]. PODXL plays a criticalrole in cancer development, and PODXL-knockout (KO) mice display lethality before
birth [7]. Importantly, PODXL is aberrantly expressed in a broad range of human tumors,
including oral squamous cell carcinoma [8], colorectal cancer [9], renal cell carcinoma [10],Int. J. Mol. Sci. 2024, 25, 161. https://doi.org/10.3390/ijms25010161

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and pancreatic ductal adenocarcinoma (PDAC) [11]. Increased PODXL expression canhave adverse eﬀects on overall survival, disease-speciﬁc survival, and disease-free survival in several cancers [12].
PODXL regulates epithelial and tumor cell motility through interactions with the actin polymerization complexes, including the ERM (ezrin–radixin–moesin) family [13] andPDZ protein Na+/H+ exchanger regulatory factors 1 and 2 (NHERF-1/2) [14]. Ezrin and
NHERF-1/2 are adaptor proteins that have diverse binding partners and facilitate the interaction of PODXL with the cytoskeletons of tumor cells [12]. The interactions between
PODXL, ezrin, and NHERF-1/2 have been reported to activate intracellular signaling including Rac1, RhoA, Cdc42, mitogen-activated protein kinase, and phosphatidylinositol3 kinase pathways to promote motility [12]. PODXL has been shown in gain- and loss-offunction studies to play an essential role in tumor progression by promoting migration,
invasiveness, stemness, and metastasis in a variety of cancer cells [15]. Therefore, PODXL
has acquired increasing attention as a target of tumor immunotherapy.
Preclinical studies have shown the promising eﬃcacy of anti-PODXL monoclonal antibodies (mAbs). A core protein-binding anti-PODXL mAb (PODO83/PODOC1) suppressed MDA-MB-231 xenograft growth and blocked metastasis to the lung [16]. Ourgroup also established an anti-PODXL mAb, PcMab-47 (mouse IgG1, kappa), which is useful for ﬂow cytometry and immunohistochemistry [17]. We engineered PcMab-47 into 47mG2a, a mouse IgG2a-type mAb, to add antibody-dependent cellular cytotoxicity (ADCC)
and further developed 47-mG2a-f, a core fucose-deﬁcient type of 47-mG2a to augment its
ADCC. Both 47-mG2a and 47-mG2a-f showed antitumor activity against PODXL-expressing oral squamous cell carcinoma-bearing mouse models [18]. Furthermore, we developed
a mouse–human chimeric PcMab-47, chPcMab-47, and showed its antitumor eﬃcacy
against PODXL-positive colorectal adenocarcinomas [19].
Nevertheless, the further development of anti-PODXL mAbs targeting tumors has
been hampered by concerns about possible toxicities to normal vascular/lymphatic endothelial cells [5] and kidney podocytes [6]. For the development of antibody therapy against
PODXL-positive cancers, cancer-speciﬁcity is necessary to reduce the risk of adverse effects on normal tissues. In this study, we developed a cancer-speciﬁc anti-PODXL mAb,
PcMab-6 (IgG1, kappa), by screening more than one hundred hybridoma clones. We engineered PcMab-6 into a mouse IgG2a-type (PcMab-6-mG2a) and a humanized IgG1-type(humPcMab-6) mAb and further produced the core fucose-deﬁcient types (PcMab-6mG2a-f and humPcMab-6-f, respectively) to potentiate ADCC. This technique was clinically applied to mogamulizumab (Poteligeo®), a defucosylated antibody targeting CCR4[20]. We then examined the antitumor activity against mouse xenograft models of PDAC.
2. Results
2.1. Development of an Anti-PODXL CasMab, PcMab-6
Using recombinant PODXL derived from glioblastoma LN229 as an antigen, we developed more than one hundred clones of anti-PODXL mAbs. We further screened the reactivity to cancer and normal cells compared with a control mAb, PcMab-47 [17], using flow
cytometry. As shown in Figure 1A, a novel anti-PODXL mAb, PcMab-6 (IgG1, kappa), reacted with endogenous PODXL on a glioblastoma LN229 cell line but not with PODXL-KO
LN229 (PDIS-13). In contrast, PcMab-47 showed stronger reactivity to LN229 but not to
PDIS-13 (Figure 1A). Similar reactivities were also observed in PDAC cell lines, including
MIA PaCa-2, Capan-2, and PK-45H (Figure 1B). PODXL is highly expressed in vascular andlymphatic endothelial cells [5]. As shown in Figure 1C, PcMab-47 stained the PODXL of alymphatic endothelial cell (LEC), HDMVEC/TERT164-B. In contrast, PcMab-6 did not reactwith it (Figure 1C), indicating that PcMab-6 shows cancer-specific reactivity.

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Figure 1. The reactivity of anti-PODXL mAbs (PcMab-6 and PcMab-47) against cancer and normal
cells using ﬂow cytometry. (A) LN229 and PODXL-KO LN229 (PDIS-13) cells were treated with 10µg/mL of PcMab-6 (red line), PcMab-47 (red line), or blocking buﬀer (black line). (B) Pancreaticcancer MIA PaCa-2, Capan-2, and PK-45H cells were treated with 10 µg/mL of PcMab-6 (red line),
PcMab-47 (red line), or blocking buﬀer (black line). (C) Lymphatic endothelial HDMVEC/TERT164B cells were treated with 10 µg/mL of PcMab-6 (red line), PcMab-47 (red line), or blocking buﬀer(black line). Then, cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG.

2.2. Production of PcMab-6-mG2a-f, a Defucosylated Mouse IgG2a-Type PcMab-6
We engineered a mouse IgG2a-type PcMab-6 (PcMab-6-mG2a) by fusing the VH chainof PcMab-6 with the CH chain of mouse IgG2a (Figure 2A). We further produced the corefucose-deleted version, PcMab-6-mG2a-f, using Fut8-deﬁcient ExpiCHO-S (BINDS-09)
cells (Figure 2A). We then investigated whether PcMab-6-mG2a-f could exert ADCC
against pancreatic cancer cells in the presence of BALB/c nude mice-derived splenocytes
as an eﬀector. As shown in Figure 2B, PcMab-6-mG2a-f showed ADCC against MIA PaCa2 (66.0% vs. 24.2% cytotoxicity of control mIgG2a, p < 0.01), Capan-2 (78.9% vs. 32.2% cytotoxicity of control mIgG2a, p < 0.05), and PK-45H (57.2% vs. 41.3% cytotoxicity of control
mIgG2a, p < 0.05). Control mIgG2a is a reference mAb that does not recognize PODXL.
We then examined whether PcMab-6-mG2a-f could exhibit complement-dependent
cellular cytotoxicity (CDC) against pancreatic cancer cells. As shown in Figure 2C, PcMab6-mG2a-f elicited a higher degree of CDC against MIA PaCa-2 (82.9% vs. 14.8% cytotoxicity
of control mIgG2a, p < 0.01), Capan-2 (74.4% vs. 49.0% cytotoxicity of control mIgG2a, p <0.05), and PK-45H (64.0% vs. 35.5% cytotoxicity of control mIgG2a, p < 0.01). These results

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demonstrated that PcMab-6-mG2a-f exerted higher levels of ADCC and CDC against pancreatic cancer cells, which depend on the recognition of PODXL.

Figure 2. ADCC and CDC via PcMab-6-mG2a-f. (A) The VH cDNA of PcMab-6 (mouse IgG1) and theCH of mouse IgG2a were cloned into a vector. The VL and CL cDNAs of PcMab-6 were cloned intoanother vector. To generate a core-fucose-deﬁcient mouse IgG2a mAb (PcMab-6-mG2a-f), the antibody expression vectors were transfected into BINDS-09 (Fut8-knocked-out ExpiCHO-S cells). (B)
ADCC induced by PcMab-6-mG2a-f or control mouse IgG2a (mIgG2a) against MIA PaCa-2, Capan-2,
and PK-45H cells. (C) CDC induced by PcMab-6-mG2a-f or control mIgG2a against MIA PaCa-2,

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Capan-2, and PK-45H cells. Values are shown as mean ± SEM. Asterisks indicate statistical signiﬁcance (** p < 0.01, * p < 0.05; Welch’s t-test).

2.3. Antitumor Activity of PcMab-6-mG2a-f against Pancreatic Cancer Xenografts
Following the inoculation of MIA PaCa-2 or Capan-2, PcMab-6-mG2a-f or control
mIgG was intraperitoneally injected into pancreatic cancer xenograft-bearing mice ondays 7, 14, and 21. On days 7, 9, 14, 16, 21, 23, and 28 after the inoculation, the tumor
volume was measured. The PcMab-6-mG2a-f administration resulted in a signiﬁcant reduction in MIA PaCa-2 xenografts on days 16 (p < 0.01), 21 (p < 0.01), 23 (p < 0.01), and 28(p < 0.01) compared with that of control mIgG (Figure 3A). A signiﬁcant reduction was
also observed in the Capan-2 xenograft on days 21 (p < 0.05), 23 (p < 0.01), and 28 (p < 0.01)(Figure 3B).
In the PK-45H xenograft, PcMab-6-mG2a-f or control mIgG was intraperitoneally injected into the mice on days 4, 11, and 18. On days 4, 6, 11, 13, 18, 20, and 25 after the inoculation, the tumor volume was measured. The PcMab-6-mG2a-f administration resulted in asignificant reduction in PK-45H xenografts on days 11 (p < 0.05), 13 (p < 0.01), 18 (p < 0.01),
20 (p < 0.01), and 25 (p < 0.01) compared with that of control mIgG (Figure 3C).
A signiﬁcant reduction in xenograft weight caused by PcMab-6-mG2a-f was observedin MIA PaCa-2 (60% reduction; p < 0.01; Figure 3D), Capan-2 (33% reduction; p < 0.05;
Figure 3E), and PK-45H (27% reduction; p < 0.05; Figure 3F). The MIA PaCa-2 and Capan2 xenografts that were resected from mice on day 28 are demonstrated in Figure 3G,H,
respectively. The PK-45H xenografts that were resected from mice on day 25 are demonstrated in Figure 3I.
Body weight loss and skin disorders were not observed in the xenograft-bearing mice
(Figure 3J–L). The mice on day 28 (MIA PaCa-2 and Capan-2) and day 25 (PK-45H) are
shown in Supplementary Figure S1.

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Figure 3. Antitumor activity of PcMab-6-mG2a-f against pancreatic cancer xenografts. (A–C) MIA
PaCa-2 (A), Capan-2 (B), and PK-45H (C) cells were subcutaneously injected into BALB/c nude mice(day 0). In total, 100 µg of PcMab-6-mG2a-f or control mouse IgG (mIgG) were intraperitoneally injected into each mouse on day 4 (PK-45H) or day 7 (MIA PaCa-2 and Capan-2). Additional antibodies were injected on days 11 and 18 (PK-45H) or days 14 and 21 (MIA PaCa-2 and Capan-2). The
tumor volume is represented as the mean ± SEM. ** p < 0.01, * p < 0.05 (ANOVA and Sidak’s multiple
comparisons test). (D–F) The mice were euthanized on day 25 (PK-45H) or day 28 (MIA PaCa-2 andCapan-2) after cell implantation. The tumor weights of MIA PaCa-2 (D), Capan-2 (E), and PK-45H(F) xenografts were measured. Values are presented as the mean ± SEM. ** p < 0.01, * p < 0.05 (Welch’s
t-test). (G–I) MIA PaCa-2 (G), Capan-2 (H), and PK-45H (I) xenograft tumors (scale bar, 1 cm). (J–L) Body weights of MIA PaCa-2 (J), Capan-2 (K), and PK-45H (L) xenograft-bearing mice treatedwith control mIgG or PcMab-6-mG2a-f. n.s., not signiﬁcant.

2.4. Production of a Humanized and Defucosylated Antibody, humPcMab-6-f
We next engineered a humanized PcMab-6 (humPcMab-6) by fusing the VH and VL
complementarity determining regions (CDRs) of PcMab-6 with the CH and CL chains ofhuman IgG1, respectively (Figure 4A). We further produced the core-fucose-deﬁcient version of humPcMab-6 (humPcMab-6-f), as described above, and investigated whether
humPcMab-6-f can detect PODXL-expressed cancer cells. The humPcMab-6-f showed
moderate reactivity to LN229 (Figure 4B) and PDAC cell lines, such as MIA PaCa-2, Capan-2, and PK-45H (Figure 4C), and showed very low reactivity to HDMVEC/TERT164-B

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7 of 16(Figure 4D). The humPcMab-6-f did not recognize PODXL-KO LN229 (PDIS-13), as shownin Figure 4B.

Figure 4. Establishment of the humanized and defucosylated antibody humPcMab-6-f. (A) The complementarity determining region (CDR) of PcMab-6 VH, the frame sequences of VH in human Ig, andthe CH of human IgG1 were cloned into a vector. The CDR of PcMab-6 VL, the frame sequences of VL
in human Ig, and the CL of the human kappa chain were cloned into another vector. To generate acore-fucose-deﬁcient form (humPcMab-6-f), the antibody expression vectors were transfected into
BINDS-09 cells. (B) LN229 and PODXL-KO LN229 (PDIS-13) cells were treated with 10 µg/mL of
humPcMab-6-f (red line) or blocking buﬀer (black line). (C) MIA PaCa-2, Capan-2, and PK-45H cells
were treated with 10 µg/mL of humPcMab-6-f (red line) or blocking buﬀer (black line). (D)HDMVEC/TERT164-B cells were treated with 10 µg/mL of humPcMab-6-f (red line) or blocking
buﬀer (black line). Then, cells were treated with FITC-conjugated anti-human IgG.

2.5. ADCC and CDC Caused by humPcMab-6-f against Pancreatic Cancers
We next examined ADCC caused by humPcMab-6-f against pancreatic cancer cells in
the presence of human natural killer (NK) cells as an eﬀector. As shown in Figure 5A,
humPcMab-6-f showed ADCC against MIA PaCa-2 (61.5% vs. 13.6% cytotoxicity of

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control hIgG, p < 0.05), Capan-2 (56.2% vs. 16.2% cytotoxicity of control hIgG, p < 0.01),
and PK-45H (72.9% vs. 16.7% cytotoxicity of control hIgG, p < 0.01). Control hIgG is a reference mAb that does not recognize PODXL.
We then investigated CDC caused by humPcMab-6-f against pancreatic cancer cells.
As shown in Figure 5B, humPcMab-6-f elicited a higher degree of CDC against MIA PaCa2 (74.7% vs. 39.6% cytotoxicity of control hIgG, p < 0.05), Capan-2 (46.3% vs. 7.3% cytotoxicity of control hIgG, p < 0.01), and PK-45H (60.8% vs. 13.3% cytotoxicity of control hIgG,
p < 0.05). These results demonstrated that humPcMab-6-f exerted very high levels of
ADCC and CDC against pancreatic cancer cells, which depend on the recognition of
PODXL.

Figure 5. ADCC and CDC caused by humPcMab-6-f against pancreatic cancers. (A) ADCC inducedby humPcMab-6-f or control human IgG (hIgG) against MIA PaCa-2, Capan-2, and PK-45H cells.
(B) CDC induced by humPcMab-6-f or control hIgG against MIA PaCa-2, Capan-2, and PK-45H
cells. Values are shown as mean ± SEM. Asterisks indicate statistical signiﬁcance (** p < 0.01, * p <0.05; Welch’s t-test).

2.6. Antitumor Activity of humPcMab-6-f against Pancreatic Cancer Xenografts
Following the inoculation of the pancreatic cancers, humPcMab-6-f or control hIgG
was intraperitoneally injected into pancreatic cancer xenograft tumor-bearing mice ondays 7 and 14. Human NK cells were also injected around the tumors on days 7 and 14.
On days 7, 10, 14, 17, and 21 after the inoculation, the tumor volume was measured. The
humPcMab-6-f administration resulted in a signiﬁcant reduction in MIA PaCa-2 xenografts on days 10 (p < 0.05), 14 (p < 0.01), 17 (p < 0.01), and 21 (p < 0.01) compared with thatof hIgG (Figure 6A). A signiﬁcant reduction was also observed in the Capan-2 xenografton days 17 (p < 0.05) and 21 (p < 0.01) (Figure 6B) and in PK-45H xenograft on days 10 (p <0.01), 14 (p < 0.01), 17 (p < 0.01), and 21 (p < 0.01) (Figure 6C).

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A signiﬁcant reduction in xenograft weight caused by humPcMab-6-f was observedin MIA PaCa-2 (30% reduction; p < 0.05; Figure 6D), Capan-2 (31% reduction; p < 0.01;
Figure 6E), and PK-45H (26% reduction; p < 0.05; Figure 6F). The MIA PaCa-2, Capan-2,
and PK-45H xenografts that were resected from mice on day 21 are demonstrated in Figure 6G,H,I, respectively.
Body weight loss and skin disorders were not observed in the xenograft-bearing mice
(Figure 6J–L). The mice on day 21 are shown in Supplementary Figure S2.

Figure 6. Antitumor activity of humPcMab-6-f against pancreatic cancer xenografts. (A–C) MIA
PaCa-2 (A), Capan-2 (B), and PK-45H (C) cells were subcutaneously injected into BALB/c nude mice(day 0). In total, 100 µg of humPcMab-6-f or control hIgG were intraperitoneally injected into eachmouse on day 7. Additional antibodies were injected on day 14. Human NK cells were also injectedaround the tumors on days 7 and 14. The tumor volume is represented as the mean ± SEM. ** p <0.01, * p < 0.05 (ANOVA and Sidak’s multiple comparisons test). (D–F) The mice were euthanized
on day 21 after cell implantation. The tumor weights of the MIA PaCa-2 (D), Capan-2 (E), and PK45H (F) xenografts were measured. Values are presented as the mean ± SEM. ** p < 0.01, * p < 0.05
(Welch’s t-test). (G–I) MIA PaCa-2 (G), Capan-2 (H), and PK-45H (I) xenograft tumors (scale bar, 1cm). (J–L) Body weights of MIA PaCa-2 (J), Capan-2 (K), and PK-45H (L) xenograft-bearing micetreated with control hIgG or humPcMab-6-f. n.s., not signiﬁcant.

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3. Discussion
Pancreatic cancer has become the third leading cause of death in men and women
combined in the United States as of 2023 [21]. PDAC is the most common type of pancreatic cancer and exhibits an extremely poor prognosis with a 5-year survival rate of approximately 10% [22]. The four most common oncogenic events, including KRAS, CDKN2A,
SMAD4, and TP53, play critical roles in cancer development [23,24]. In contrast, PDAC isa heterogenous tumor with various histologies [25], heterogenous molecular landscapes[26], and clinical outcomes. Therefore, the identiﬁcation of early diagnostic markers andtherapeutic targets in each group is desired. In this study, we provided evidence that
PODXL could be a promising target for antibody-based therapy, especially PcMab-6 (Figure 1) and its derivatives, including PcMab-6-mG2a-f (Figures 2 and 3) and humPcMab-6f (Figures 4–6).
High PODXL expression is signiﬁcantly associated with worse overall survival andis predictive of shorter overall survival in multiple cancers, especially PDAC [27]. Furthermore, PODXL is upregulated during epithelial–mesenchymal transition [28] and plays a
key role in the extravasation of mesenchymal PDAC cells [29]. PODXL enhances the extravasation through direct binding to ezrin, a cytoskeletal linker protein, which enables
the transition of tumor cells from a non-polarized, rounded cell shape to an invasive extravasation-competent morphology [29]. These results suggest that PODXL mediates theprocess of extravasation during tumor metastasis. Although the requirement of PODXL
ligands such as E-selectin [30] in endothelial cells is unknown, it would be interesting to
see whether PcMab-6 aﬀects PODXL ligand interactions, tumor cell extravasation, andmetastasis.
We developed a PODXL-targeting CasMab in cancer cells by screening more than
one hundred clones of anti-PODXL mAbs. PcMab-6 can recognize PDAC cells but notnormal LECs in ﬂow cytometry (Figure 1). We previously developed CasMabs against
podoplanin (PDPN) [31]. The CasMabs were selected via ﬂow cytometry and immunohistochemistry and exhibited reactivity with cancer cells but reduced reactivity with normal cells [32]. An anti-PDPN CasMab (clone LpMab-2), which has been applied to chimeric antigen receptor (CAR)-T therapy in mice preclinical models [33], recognizes theglycopeptide from Thr55 to Leu64 of PDPN [32]. Another CasMab, LpMab-23, recognizes
a peptide from Gly54 to Leu64 of PDPN [34].
The minimum epitopes of one of the anti-PODXL non-CasMabs (clone PcMab-47)
and another anti-PODXL CasMab (clone PcMab-60 [35]) were determined to be the peptide sequence, including Asp207, His208, Leu209, and Met210, of PODXL [36] and the
peptide sequence, including Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, andPro116, of PODXL [37], respectively. In contrast, the epitope of PcMab-6 has not been
identiﬁed yet. The identiﬁcation of the PcMab-6 epitope is essential for understanding themechanism of cancer-speciﬁc recognition. Furthermore, recognition by PcMab-6 in ﬂow
cytometry was lower than that of PcMab-47 (Figure 1), which can be attributed to the possibility that the epitope sequence is partially exposed or modiﬁed in cancer cells but not
in normal cells in living cells. The CasMab selection strategy can contribute to the development of novel mAbs against a variety of antigens.
Glycosylation in tumors is frequently altered [38]. Anti-PODXL mAbs, which recognize tumor-speciﬁc glyco-epitopes on PODXL but do not react with PODXL expressed on
normal cells, have been developed [39]. One of these mAbs, PODO447, exhibits exquisite
speciﬁcity for a tumor glycoform of PODXL but lacks reactivity with normal adult humantissue. Using an array of glycosylation defective cell lines, the epitope of PODO447 wasrevealed to be an O-linked core 1 glycan presented in the context of the PODXL peptide
backbone [39]. Furthermore, PODO447 coupled with monomethyl auristatin E (MMAE)
showed eﬃcacy in targeting human pancreatic and ovarian tumor xenografts in mousemodels [40].
All therapeutic mAbs exhibit adverse eﬀects, probably because of the recognition of
antigens expressed in normal cells. The majority of patients treated with anti-EGFR

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therapeutic mAbs experience dermatological disorders, which aﬀect their quality of life
and adherence to the therapy [41]. The major adverse eﬀect associated with anti-HER2
therapeutic mAbs is cardiotoxicity [42]. Since PODXL is expressed in normal adult tissues,
including vascular/lymphatic endothelial cells [5] and kidney podocytes [6], the evaluation of the in vivo toxicity of PcMab-6 derivatives is essential. We investigated this toxicity
against cynomolgus monkeys. The ﬁrst protocol is one intravenous injection of 20 mg/kg
of the mouse–human chimeric and core-fucose-deleted antibody of PcMab-6 (chPcMab-6f) and observation for one week. The second protocol is four intravenous injections of 10
mg/kg of chPcMab-6-f (every week) and observation for one month. We conﬁrmed that
chPcMab-6-f did not show any toxicities against cynomolgus monkeys in either protocol(Supplementary Figure S3).
CARs are synthetic modular proteins that redirect immune cell reactivity toward atarget of interest. This platform has shown substantial clinical eﬀects against B cell andplasma cell malignancies and the potential to expand its application to solid tumors [43].
The optimal epitope for the PODXL-targeting CAR has not yet been evaluated. However,
it would be worthwhile to investigate the cancer speciﬁcity of the PcMab-6 single-chain
variable fragment and the eﬃcacy of CAR-T therapy against PODXL-positive tumors.
4. Materials and Methods
4.1. Cell Lines
A mouse myeloma cell line, P3X63Ag8U.1 (P3U1); a human glioblastoma cell line
(LN229); and a PDAC cell line (Capan-2) were obtained from the American Type Culture
Collection (Manassas, VA, USA). Two PDAC cell lines (MIA PaCa-2 and PK-45H) were obtained from the Cell Resource Center for the Biomedical Research Institute of Development,
Aging, and Cancer of Tohoku University (Miyagi, Japan). The LN229/PODXL ectodomain(LN229/PODXLec) was produced in our previous study [17]. PODXL-KO LN229 (PDIS-13)
was generated as described previously [19]. LN229, PDIS-13, LN229/PODXLec, and MIA
PaCa-2 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque,Inc., Kyoto, Japan). Capan-2 was cultured in McCoy’s 5A medium (Cytiva, Tokyo, Japan).
P3U1 and PK-45H were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium(Nacalai Tesque, Inc., Kyoto, Japan). All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA, USA), 100
units/mL of penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of amphotericin B(Nacalai Tesque, Inc.). A lymphatic endothelial cell line (HDMVEC/TERT164-B) was purchased from EVERCYTE (Vienna, Austria) and was cultured in an Endopan MV kit (PAN
Biotech, Bayern, Germany) supplemented with G418. All cells were cultured at 37 ◦C in ahumidified atmosphere containing 5% CO2 and 95% air.
4.2. Animals
For the establishment of mAbs against PODXL, the animal experiment was approvedby the Animal Care and Use Committee of Tohoku University. For the ADCC assay andantitumor activity in mouse xenograft models, the animal experiments were approved by
the Institutional Committee for Experiments of the Institute of Microbial Chemistry. 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.
4.3. Hybridoma Production
Female BALB/c mice (CLEA, Tokyo, Japan) were immunized with puriﬁed PODXLec(100 µg) [17] together with Imject Alum (Thermo Fisher Scientiﬁc Inc.) via intraperitoneal
injection. After several additional immunizations of PODXLec, the spleen cells were harvested and fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN,
USA). Hybridomas were grown in RPMI-1640 medium, including L-glutamine with hypoxanthine, aminopterin, and thymidine (HAT) selection medium supplement (Thermo

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Fisher Scientiﬁc Inc.). Culture supernatants were screened using enzyme-linked immunosorbent assay (ELISA) for binding to PODXLec.
4.4. ELISAPODXLec was immobilized on Nunc Maxisorp 96-well immunoplates (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.), theplates were incubated with culture supernatant followed by 1:2000 diluted peroxidaseconjugated anti-mouse immunoglobulins (Agilent Technologies, Inc., Santa Clara, CA,
USA). The enzymatic reaction was produced with a 1-Step Ultra TMB-ELISA (Thermo
Fisher Scientiﬁc, Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA).
4.5. Antibodies
A mouse anti-PODXL mAb, PcMab-47 (IgG1, kappa), was developed as described
previously [17]. Normal mouse IgG (mIgG) was purchased from FUJIFILM Wako Pure
Chemical Corporation (Osaka, Japan). Mouse IgG2a (mIgG2a) and normal human IgG(hIgG) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). To generate
PcMab-6-mG2a, the VH cDNA of PcMab-6 and the CH of mouse IgG2a were subcloned intoa pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation), and the VL and CL
cDNAs of PcMab-6 were subcloned into a pCAG-Neo vector (FUJIFILM Wako Pure
Chemical Corporation). To generate a humanized PcMab-6 (humPcMab-6), the CDR of
PcMab-6 VH, the frame sequences of VH in human Ig, and the CH of human IgG1 werecloned into the pCAG-Neo vector. The CDR of PcMab-6 VL, the frame sequences of VL in
human Ig, and the CL of the human kappa chain were cloned into the pCAG-Ble vector.
To generate PcMab-6-mG2a-f and humPcMab-6-f, antibody expression vectors were alsotransfected into BINDS-09 (Fut8-knocked-out ExpiCHO-S cells) using the ExpiCHO Expression System. PcMab-6, PcMab-6-mG2a-f, and humPcMab-6-f were puriﬁed using Protein G-Sepharose (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).
4.6. Flow Cytometry
Cell lines were harvested via brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). After washing with 0.1% BSA in PBS(blocking buﬀer), the cells were treated with primary 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, USA) or ﬂuorescein isothiocyanate (FITC)-conjugated anti-human IgG (1:2000; Sigma-Aldrich Corp.). Fluorescence data were collected using an SA3800
Cell Analyzer (Sony Corp., Tokyo, Japan).
4.7. ADCC
The ADCC activity of PcMab-6-mG2a-f was measured as follows. In brief, eﬀector
cells were obtained from the spleens of female BALB/c nude mice (Jackson Laboratory
Japan, Inc., Kanagawa, Japan). We labeled target cells (MIA PaCa-2, Capan-2, and PK45H) using 10 µg/mL of Calcein AM (Thermo Fisher Scientiﬁc, Inc.), plated them on 96well plates (1 × 104 cells/well), and mixed them with the eﬀector cells (eﬀector-to-target
ratio, 100:1) with 100 µg/mL of control mIgG2a or PcMab-6-mG2a-f. After a 4.5 h incubation
at 37 °C, the Calcein release into the medium was measured using a microplate reader(Power Scan HT; BioTek Instruments, Inc., Winooski, VT, USA).
The ADCC activity of humPcMab-6-f was measured as follows. The Calcein AM-labeled target cells (MIA PaCa-2, Capan-2, and PK-45H) were mixed with human NK cells(Takara Bio, Inc., Shiga, Japan; eﬀector-to-target ratio, 50:1) with 100 µg/mL of control

Int. J. Mol. Sci. 2024, 25, 161

13 of 16hIgG or humPcMab-6-f. The Calcein release into the medium was measured after a 4.5 hincubation.
Cytolyticity (% lysis) was determined: % lysis is calculated as (E − S)/(M − S) × 100,
where “E” denotes the ﬂuorescence in the eﬀector and target cell cultures, “S” denotes the
spontaneous ﬂuorescence of only target cells, and “M” denotes the maximum ﬂuorescence
after treatment with a lysis buﬀer (10 mM Tris-HCl (pH 7.4), 10 mM EDTA, and 0.5%
Triton X-100). All data are shown as mean ± standard error of the mean (SEM). Welch’s ttest was used for the statistical analyses.
4.8. CDC
The Calcein AM-labeled target cells (MIA PaCa-2, Capan-2, and PK-45H) 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 mIgG2a, PcMab6-mG2a-f, control hIgG, or humPcMab-6-f. Following incubation for 4.5 h at 37 °C, the Calcein release into the medium was measured, as described above.
4.9. Antitumor Activity of Anti-PODXL Antibodies
Female BALB/c nude mice were purchased from Jackson Laboratory Japan, Inc. The
cells (0.3 mL 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, USA). A 100 µL suspension (containing 5 × 106 cells) was injected subcutaneously into the left ﬂanks of the nude mice. In
total, 100 µg of PcMab-6-mG2a-f or control mIgG in 100 µL of PBS was injected into theperitoneal cavity of each mouse at day 4 (PK-45H) or day 7 (MIA PaCa-2 and Capan-2).
Additional antibodies were injected on days 11 and 18 (PK-45H) or days 14 and 21 (MIA
PaCa-2 and Capan-2). The mice were euthanized on day 25 (PK-45H) or day 28 (MIA
PaCa-2 and Capan-2) after the cell inoculation.
To measure the antitumor activity of humPcMab-6-f, 100 µg of humPcMab-6-f or control hIgG in 100 µL of PBS was injected into the peritoneal cavity of each tumor xenograftbearing mouse on day 7. Additional antibodies were injected on day 14. Furthermore, human NK cells (8.0 × 105 cells) were injected around the tumors on days 7 and 14. The mice
were euthanized on day 21 after the cell inoculation.
The tumor diameter was measured and the tumor volume was calculated using thefollowing formula: volume = W2 × L/2, where W is the short diameter, and L is the long
diameter. All data are expressed as the mean ± SEM. Statistical analysis was performed
using ANOVA with Sidak’s post hoc test. p < 0.05 was considered statistically signiﬁcant.
Supplementary Materials: The supporting information
https://www.mdpi.com/article/10.3390/ijms25010161/s1.

can

be

downloadedatAuthor Contributions: H.S., T.O. and T.T. performed the experiments. M.K.K. and Y.K. designed
the experiments. H.S. and Y.K. analyzed the data. H.S. and Y.K. wrote the manuscript. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was supported in part by the 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) grantnos. 22K06995 (to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Institutional Review Board Statement: Animal experiments were approved by the Animal Careand Use Committee of Tohoku University (permit number: 2019NiA-001) and the Institutional Committee for Experiments of the Institute of Microbial Chemistry (approval nos. 2022-010, 2022-017,
2022-021, and 2022-045).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article and Supplementary Material.

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Acknowledgments: The authors would like to thank Shun-ichi Ohba and Akiko Harakawa (Institute of 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.

References
1.

2.
3.

4.

5.
6.
7.
8.
9.

10.
11.

12.
13.
14.
15.
16.

17.

18.

19.
20.
21.

Doyonnas, R.; Nielsen, J.S.; Chelliah, S.; Drew, E.; Hara, T.; Miyajima, A.; McNagny, K.M. Podocalyxin is a CD34-related marker
of murine hematopoietic stem cells and embryonic erythroid cells. Blood 2005, 105, 4170–4178. https://doi.org/10.1182/blood2004-10-4077.
Nielsen, J.S.; McNagny, K.M. Novel functions of the CD34 family. J. Cell Sci. 2008, 121, 3683–3692. https://doi.org/10.1242/jcs.037507.
Choo, A.B.; Tan, H.L.; Ang, S.N.; Fong, W.J.; Chin, A.; Lo, J.; Zheng, L.; Hentze, H.; Philp, R.J.; Oh, S.K.; et al. Selection against
undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1. Stem Cells 2008,
26, 1454–1463. https://doi.org/10.1634/stemcells.2007-0576.
McNagny, K.M.; Pettersson, I.; Rossi, F.; Flamme, I.; Shevchenko, A.; Mann, M.; Graf, T. Thrombomucin, a novel cell surface
protein that defines thrombocytes and multipotent hematopoietic progenitors. J. Cell Biol. 1997, 138, 1395–1407.
https://doi.org/10.1083/jcb.138.6.1395.
Kriehuber, E.; Breiteneder-Geleff, S.; Groeger, M.; Soleiman, A.; Schoppmann, S.F.; Stingl, G.; Kerjaschki, D.; Maurer, D.
Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized celllineages. J. Exp. Med. 2001, 194, 797–808. https://doi.org/10.1084/jem.194.6.797.
Kerjaschki, D.; Sharkey, D.J.; Farquhar, M.G. Identification and characterization of podocalyxin--the major sialoprotein of therenal glomerular epithelial cell. J. Cell Biol. 1984, 98, 1591–1596. https://doi.org/10.1083/jcb.98.4.1591.
Doyonnas, R.; Kershaw, D.B.; Duhme, C.; Merkens, H.; Chelliah, S.; Graf, T.; McNagny, K.M. Anuria, omphalocele, and perinatal
lethality in mice lacking the CD34-related protein podocalyxin. J. Exp. Med. 2001, 194, 13–27. https://doi.org/10.1084/jem.194.1.13.
Lin, C.W.; Sun, M.S.; Wu, H.C. Podocalyxin-like 1 is associated with tumor aggressiveness and metastatic gene expression inhuman oral squamous cell carcinoma. Int. J. Oncol. 2014, 45, 710–718. https://doi.org/10.3892/ijo.2014.2427.
Larsson, A.; Johansson, M.E.; Wangefjord, S.; Gaber, A.; Nodin, B.; Kucharzewska, P.; Welinder, C.; Belting, M.; Eberhard, J.;
Johnsson, A.; et al. Overexpression of podocalyxin-like protein is an independent factor of poor prognosis in colorectal cancer.
Br. J. Cancer 2011, 105, 666–672. https://doi.org/10.1038/bjc.2011.295.
Hsu, Y.H.; Lin, W.L.; Hou, Y.T.; Pu, Y.S.; Shun, C.T.; Chen, C.L.; Wu, Y.Y.; Chen, J.Y.; Chen, T.H.; Jou, T.S. Podocalyxin EBP50
ezrin molecular complex enhances the metastatic potential of renal cell carcinoma through recruiting Rac1 guanine nucleotide
exchange factor ARHGEF7. Am. J. Pathol. 2010, 176, 3050–3061. https://doi.org/10.2353/ajpath.2010.090539.
Ney, J.T.; Zhou, H.; Sipos, B.; Büttner, R.; Chen, X.; Klöppel, G.; Gütgemann, I. Podocalyxin-like protein 1 expression is useful
to differentiate pancreatic ductal adenocarcinomas from adenocarcinomas of the biliary and gastrointestinal tracts. Hum. Pathol.
2007, 38, 359–364. https://doi.org/10.1016/j.humpath.2006.08.025.
Le Tran, N.; Wang, Y.; Nie, G. Podocalyxin in Normal Tissue and Epithelial Cancer. Cancers 2021, 13, 2863.
https://doi.org/10.3390/cancers13122863.
Sizemore, S.; Cicek, M.; Sizemore, N.; Ng, K.P.; Casey, G. Podocalyxin increases the aggressive phenotype of breast and prostate cancer
cells in vitro through its interaction with ezrin. Cancer Res. 2007, 67, 6183–6191. https://doi.org/10.1158/0008-5472.Can-06-3575.
Meder, D.; Shevchenko, A.; Simons, K.; Füllekrug, J. Gp135/podocalyxin and NHERF-2 participate in the formation of apreapical domain during polarization of MDCK cells. J. Cell Biol. 2005, 168, 303–313. https://doi.org/10.1083/jcb.200407072.
Tamayo-Orbegozo, E.; Amo, L.; Díez-García, J.; Amutio, E.; Riñón, M.; Alonso, M.; Arana, P.; Maruri, N.; Larrucea, S. Emerging
Role of Podocalyxin in the Progression of Mature B-Cell Non-Hodgkin Lymphoma. Cancers 2020, 12, 396.
https://doi.org/10.3390/cancers12020396.
Snyder, K.A.; Hughes, M.R.; Hedberg, B.; Brandon, J.; Hernaez, D.C.; Bergqvist, P.; Cruz, F.; Po, K.; Graves, M.L.; Turvey, M.E.;
et al. Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy. Breast Cancer
Res. 2015, 17, 46. https://doi.org/10.1186/s13058-015-0562-7.
Ogasawara, S.; Kaneko, M.K.; Yamada, S.; Honma, R.; Nakamura, T.; Saidoh, N.; Yanaka, M.; Yoshida, K.; Fujii, Y.; Kato, Y.
PcMab-47: Novel Antihuman Podocalyxin Monoclonal Antibody for Immunohistochemistry. Monoclon. Antib. Immunodiagn.
Immunother. 2017, 36, 50–56. https://doi.org/10.1089/mab.2017.0008.
Itai, S.; Ohishi, T.; Kaneko, M.K.; Yamada, S.; Abe, S.; Nakamura, T.; Yanaka, M.; Chang, Y.W.; Ohba, S.I.; Nishioka, Y.; et al.
Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models oforal squamous cell carcinoma. Oncotarget 2018, 9, 22480–22497. https://doi.org/10.18632/oncotarget.25132.
Kaneko, M.K.; Kunita, A.; Yamada, S.; Nakamura, T.; Yanaka, M.; Saidoh, N.; Chang, Y.W.; Handa, S.; Ogasawara, S.; Ohishi,T.; et al. Antipodocalyxin Antibody chPcMab-47 Exerts Antitumor Activity in Mouse Xenograft Models of Colorectal
Adenocarcinomas. Monoclon. Antib. Immunodiagn. Immunother. 2017, 36, 157–162. https://doi.org/10.1089/mab.2017.0020.
Subramaniam, J.M.; Whiteside, G.; McKeage, K.; Croxtall, J.C. Mogamulizumab: First global approval. Drugs 2012, 72, 1293–
1298. https://doi.org/10.2165/11631090-000000000-00000.
Siegel, R.L.; Miller, K.D.; Wagle, N.S.; Jemal, A. Cancer statistics, 2023. CA Cancer J. Clin. 2023, 73, 17–48.
https://doi.org/10.3322/caac.21763.

Int. J. Mol. Sci. 2024, 25, 161

22.
23.

24.

25.
26.

27.

28.
29.

30.

31.
32.
33.
34.

35.

36.
37.

38.
39.
40.

41.

15 of 16

Kamisawa, T.; Wood, L.D.; Itoi, T.; Takaori, K. Pancreatic cancer. Lancet 2016, 388, 73–85. https://doi.org/10.1016/s01406736(16)00141-0.
Waddell, N.; Pajic, M.; Patch, A.M.; Chang, D.K.; Kassahn, K.S.; Bailey, P.; Johns, A.L.; Miller, D.; Nones, K.; Quek, K.; et al.
Whole genomes redefine the mutational landscape of pancreatic cancer. Nature 2015, 518, 495–501.
https://doi.org/10.1038/nature14169.
Jones, S.; Zhang, X.; Parsons, D.W.; Lin, J.C.; Leary, R.J.; Angenendt, P.; Mankoo, P.; Carter, H.; Kamiyama, H.; Jimeno, A.; et al.
Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 2008, 321, 1801–1806.
https://doi.org/10.1126/science.1164368.
Taherian, M.; Wang, H.; Wang, H. Pancreatic Ductal Adenocarcinoma: Molecular Pathology and Predictive Biomarkers. Cells
2022, 11, 3068. https://doi.org/10.3390/cells11193068.
Bailey, P.; Chang, D.K.; Nones, K.; Johns, A.L.; Patch, A.M.; Gingras, M.C.; Miller, D.K.; Christ, A.N.; Bruxner, T.J.; Quinn, M.C.;
et al. Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 2016, 531, 47–52.
https://doi.org/10.1038/nature16965.
Wang, J.; Zhao, Y.; Qi, R.; Zhu, X.; Huang, C.; Cheng, S.; Wang, S.; Qi, X. Prognostic role of podocalyxin-like protein expressioninvariouscancers:A
systematic
review
andmeta-analysis.
Oncotarget
2017,8,52457–52464.
https://doi.org/10.18632/oncotarget.14199.
Chen, M.J.; Gao, X.J.; Xu, L.N.; Liu, T.F.; Liu, X.H.; Liu, L.X. Ezrin is required for epithelial-mesenchymal transition induced by
TGF-β1 in A549 cells. Int. J. Oncol. 2014, 45, 1515–1522. https://doi.org/10.3892/ijo.2014.2554.
Fröse, J.; Chen, M.B.; Hebron, K.E.; Reinhardt, F.; Hajal, C.; Zijlstra, A.; Kamm, R.D.; Weinberg, R.A. Epithelial-Mesenchymal
Transition Induces Podocalyxin to Promote Extravasation via Ezrin Signaling. Cell Rep. 2018, 24, 962–972.
https://doi.org/10.1016/j.celrep.2018.06.092.
Dallas, M.R.; Chen, S.H.; Streppel, M.M.; Sharma, S.; Maitra, A.; Konstantopoulos, K. Sialofucosylated podocalyxin is afunctional E- and L-selectin ligand expressed by metastatic pancreatic cancer cells. Am. J. Physiol. Cell Physiol. 2012, 303, C616–
C624. https://doi.org/10.1152/ajpcell.00149.2012.
Suzuki, H.; Kaneko, M.K.; Kato, Y. Roles of Podoplanin in Malignant Progression of Tumor. Cells 2022, 11, 575.
https://doi.org/10.3390/cells11030575.
Kato, Y.; Kaneko, M.K. A cancer-specific monoclonal antibody recognizes the aberrantly glycosylated podoplanin. Sci. Rep.
2014, 4, 5924. https://doi.org/10.1038/srep05924.
Chalise, L.; Kato, A.; Ohno, M.; Maeda, S.; Yamamichi, A.; Kuramitsu, S.; Shiina, S.; Takahashi, H.; Ozone, S.; Yamaguchi, J.; etal. Efficacy of cancer-specific anti-podoplanin CAR-T cells and oncolytic herpes virus G47Δ combination therapy against
glioblastoma. Mol. Ther. Oncolytics 2022, 26, 265–274. https://doi.org/10.1016/j.omto.2022.07.006.
Kaneko, M.K.; Nakamura, T.; Kunita, A.; Fukayama, M.; Abe, S.; Nishioka, Y.; Yamada, S.; Yanaka, M.; Saidoh, N.; Yoshida, K.;
et al. ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via
Antibody-Dependent Cellular Cytotoxicity. Monoclon. Antib. Immunodiagn. Immunother. 2017, 36, 104–112.
https://doi.org/10.1089/mab.2017.0014.
Kaneko, M.K.; Ohishi, T.; Kawada, M.; Kato, Y. A cancer-specific anti-podocalyxin monoclonal antibody (60-mG(2a)-f) exerts
antitumor effects in mouse xenograft models of pancreatic carcinoma. Biochem. Biophys. Rep. 2020, 24, 100826.
https://doi.org/10.1016/j.bbrep.2020.100826.
Itai, S.; Yamada, S.; Kaneko, M.K.; Kato, Y. Determination of critical epitope of PcMab-47 against human podocalyxin. Biochem.
Biophys. Rep. 2018, 14, 78–82. https://doi.org/10.1016/j.bbrep.2018.04.003.
Asano, T.; Suzuki, H.; Kaneko, M.K.; Kato, Y. Epitope Mapping of a Cancer-Specific Anti-Podocalyxin Monoclonal Antibody(PcMab-60) Using Enzyme-Linked Immunosorbent Assay and Surface Plasmon Resonance. Monoclon. Antib. Immunodiagn.
Immunother. 2021, 40, 227–232. https://doi.org/10.1089/mab.2021.0030.
Pinho, S.S.; Reis, C.A. Glycosylation in cancer: Mechanisms and clinical implications. Nat. Rev. Cancer 2015, 15, 540–555.
https://doi.org/10.1038/nrc3982.
Canals Hernaez, D.; Hughes, M.R.; Dean, P.; Bergqvist, P.; Samudio, I.; Blixt, O.; Wiedemeyer, K.; Li, Y.; Bond, C.; Cruz, E.; etal. PODO447: A novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin. J. Immunother. Cancer 2020, 8,
e001128. https://doi.org/10.1136/jitc-2020-001128.
Canals Hernaez, D.; Hughes, M.R.; Li, Y.; Mainero Rocca, I.; Dean, P.; Brassard, J.; Bell, E.M.; Samudio, I.; Mes-Masson, A.M.;
Narimatsu, Y.; et al. Targeting a Tumor-Specific Epitope on Podocalyxin Increases Survival in Human Tumor PreclinicalModels. Front. Oncol. 2022, 12, 856424. https://doi.org/10.3389/fonc.2022.856424.
Holcmann, M.; Sibilia, M. Mechanisms underlying skin disorders induced by EGFR inhibitors. Mol. Cell Oncol. 2015, 2, e1004969.
https://doi.org/10.1080/23723556.2015.1004969.

Int. J. Mol. Sci. 2024, 25, 161

42.
43.

16 of 16

Copeland-Halperin, R.S.; Liu, J.E.; Yu, A.F. Cardiotoxicity of HER2-targeted therapies. Curr. Opin. Cardiol. 2019, 34, 451–458.
https://doi.org/10.1097/hco.0000000000000637.
Labanieh, L.; Mackall, C.L. CAR immune cells: Design principles, resistance and the next generation. Nature 2023, 614, 635–648.
https://doi.org/10.1038/s41586-023-05707-3.

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