Original Article

Efficacy of cancer-specific anti-podoplanin
CAR-T cells and oncolytic herpes virus G47D
combination therapy against glioblastoma
Lushun Chalise,1,2,3 Akira Kato,11 Masasuke Ohno,4 Sachi Maeda,1 Akane Yamamichi,5 Shunichiro Kuramitsu,6Satoshi Shiina,7 Hiromi Takahashi,8 Sachiko Ozone,11 Junya Yamaguchi,1 Yukinari Kato,9,10 Yumi Rockenbach,11Atsushi Natsume,11,12 and Tomoki Todo3
1Department of Neurosurgery, Nagoya University School of Medicine, Nagoya, Japan; 2Department of Neurosurgery, Nagoya Central Hospital, Nagoya, Japan; 3Division of

Innovative Cancer Therapy, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; 4Department of Neurosurgery, Aichi
Cancer Centre Hospital, Nagoya, Japan; 5Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, USA; 6Department of
Neurosurgery, National Hospital Organization Nagoya Medical Center, Nagoya, Japan; 7East Nagoya Imaging Diagnosis Center, Nagoya, Japan; 8Department of
Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Japan; 9Department of Molecular Pharmacology, Tohoku University Graduate
School of Medicine, Sendai, Japan; 10Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan; 11The Institute of
Innovation for Future Society, Nagoya University, Nagoya, Japan; 12Department of Neurosugery, Kawamura Medical Society Hospital, Gifu, Japan

Glioblastoma is a devastating malignant brain tumor witha poor prognosis despite standard therapy. Podoplanin(PDPN), a type I transmembrane mucin-like glycoproteinthat is overexpressed in various cancers, is a potential therapeutic target for the treatment of glioblastoma. We previously reported the efﬁcacy of chimeric antigen receptor (CAR)-T cells
using an anti-pan-PDPN monoclonal antibody (mAb; NZ-1)based third-generation CAR in a xenograft mouse model. However, NZ-1 also reacted with PDPN-expressing normal cells,such as lymphatic endothelial cells, pulmonary alveolar type I
cells, and podocytes. To overcome possible on-target-off-tumor
effects, we produced a cancer-speciﬁc mAb (CasMab,
LpMab-2)-based CAR. LpMab-2 (Lp2) reacted with PDPN-expressing cancer cells but not with normal cells. In this study,
Lp2-CAR-transduced T cells (Lp2-CAR-T) speciﬁcally targeted
PDPN-expressing glioma cells while sparing the PDPN-expressing normal cells. Lp2-CAR-T also killed patient-derived
glioma stem cells, demonstrating its clinical potential against
glioblastoma. Systemic injection of Lp2-CAR-T cells inhibitedthe growth of a subcutaneous glioma xenograft model in
immunodeﬁcient mice. Combination therapy with Lp2-CART and oncolytic virus G47D, a third-generation recombinantherpes simplex virus (HSV)-1, further inhibited the tumor
growth and improved survival. These ﬁndings indicate thatthe combination therapy of Lp2-CAR-T cells and G47D may
be a promising approach to treat glioblastoma.

INTRODUCTION
Glioblastoma (GBM) is a devastating malignant brain tumor witha median survival of less than 2 years despite standard therapy that
includes surgery followed by chemotherapy and radiation.1,2
Immunotherapy, the fourth modality of anti-cancer treatment

following surgery, chemotherapy, and radiotherapy, has shownpromising results in some cancer types but does not have thesame success in the treatment of brain tumors. This calls for innovative measures to make immunotherapy more effective against
brain tumors. Chimeric antigen receptor (CAR) T cells, whichhave already proven their clinical efﬁcacy in patients with CD19positive hematological tumors, such as leukemia3,4 and lymphoma,5-7 have so far been disappointing in the ﬁght against brain
tumors, with very limited success at the cost of severe adverse
events, such as cytokine storm, pulmonary toxicity, and edema,
resulting in multiple organ dysfunction, possibly due to ontarget-off-tumor effects.8-11 Therefore, new measures to overcome
potential side effects while increasing antitumor efﬁcacy need to be
developed.
Podoplanin (PDPN), a type I transmembrane mucin-like glycoprotein, is abundant in several solid tumors including squamous cell carcinoma, malignant mesothelioma, Kaposi sarcoma, angiosarcoma,
testicular seminoma, and brain tumors.12,13 In gliomas, PDPN isoverexpressed in accordance with tumor malignancy14 and is thehighest in the mesenchymal subtype GBM.12,15,16 The potential of
PDPN has been recognized as a therapeutic target of CAR T cells
to treat brain tumors, and we previously generated a third generation

Received 23 December 2021; accepted 15 July 2022;
https://doi.org/10.1016/j.omto.2022.07.006.
Correspondence: Tomoki Todo, MD, PhD, Division of Innovative Cancer Therapy, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
E-mail: atsushi.natsume@mirai.nagoya-u.ac.jp
Correspondence: Atsushi Natsume, MD, PhD, The Institute of Innovation for
Future Society, Nagoya University, NIC Room 803, Furo-Cho, Chikusa-Ku, Nagoya
464-8601, Japan.
E-mail: toudou-nsu@umin.ac.jp

Molecular Therapy: Oncolytics Vol. 26 September 15 2022 ª 2022 The Author(s).
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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of CAR that targeted PDPN using an anti-pan-PDPN antibody,NZ-1-based single-chain variable fragments (scFvs).12,17 NZ-1-CAR
T cells showed speciﬁc efﬁcacy against PDPN-positive GBM cells
in vitro and inhibited the growth of intracranial glioma xenografts
in vivo.12 However, PDPN is also present in normal tissues, such as
the mesothelium, the lymphatic endothelium, lung type I alveolar
cells, kidney glomerular podocytes, meninges,18,19 and Purkinjecells20 in the central nervous system. This raises safety concerns
regarding PDPN-targeting therapies and necessitates the development of a better approach to speciﬁcally target cancer cells to overcome the on-target-off-tumor effect. To precisely target GBM cells,
we previously created a cancer-speciﬁc monoclonal antibody(CasMab) for human PDPN.21 A novel monoclonal antibody(mAb) clone, LpMab-2 (Lp2), recognizes the aberrantly glycosylated
cancer-type PDPN, which was extracted from a human PDPN-transduced GBM cell line. As Lp2 does not recognize PDPN-expressing
normal cells, CAR-T cell therapy based on Lp2 can also be expected
to be cancer speciﬁc.
Oncolytic viruses are promising anti-cancer therapies that have also
been tested against malignant brain tumors.22-24 They can not only
kill solid tumors directly but also elicit an antitumor immune
response.25,26 G47D is a third-generation oncolytic recombinant herpes simplex virus (HSV)-1 that was developed by adding another mutation to the genome of G207, a second-generation HSV-1.27,28 In
addition to mutations in the g34.5 and ICP6 genes, G47D has a deletion in the a47 gene that causes enhanced stimulation of antitumor
immunity and improved replication in cancer cells. G47D proved
its efﬁcacy and safety in a phase II clinical trial for GBM in Japanand was approved by the Ministry of Health, Labour and Welfare
Japan as the ﬁrst oncolytic therapy drug for malignant glioma in
the world in June 2021.
Given the fact that G47D induces a speciﬁc antitumor immune
response, a combination of CasMab-based CAR-T cells may exert amore rational antitumor therapy speciﬁc to GBM.
In this study, we established a third generation of Lp2 scFv-based
CAR that reacts with cancer-type PDPN in GBM. We report anenhanced antitumor effect when this novel CAR-T cell was used in
combination with the oncolytic virus G47D.

RESULTS
Cancer-type PDPN expression in cell lines

Immunoﬂuorescence using the antibody Lp2 demonstrated that cancer-type PDPN was positive on the cell surface of LN319, GIC0222,
and TGS01 cells. LN229 cells did not express PDPN and were therefore used as a negative control. LN229 cells transfected with human
PDPN were used as positive controls for subsequent in vitro experiments (Figure 1A). Normal mesothelial cells, MeT-5A expressing
PDPN were positive for anti-pan-PDPN mAb NZ-1, but negative
for the CasMab Lp2.21 MeT-5A cells were later used in an in vitro
cytotoxicity assay to conﬁrm the cancer speciﬁcity of CAR-T cells
based on Lp2.

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Construction of Lp2-CAR-T cells

A CasMab, Lp2 was puriﬁed from a human PDPN-transfected GBM
cell line and recognizes cancer-type PDPN.21 We constructed a lentiviral vector linked with the EF1a promoter tandemly followed by theleader sequence and Lp2-based scFvs, CD28, 4-1BB, and CD3z
(third-generation CAR) (Figure 1B). The transduction efﬁciency
was examined by ﬂow cytometry using a mouse-derived F(ab’)2biotin antibody. The percentage of transduced peripheral bloodmononuclear cells (PBMCs) with Lp2-CAR was more than 40%,and these results were reproducible (Table 1). This novel CAR-T(called Lp2-CAR-T) based on CasMab is expected to exhibit cancer-speciﬁc cytotoxicity against glioma.
In vitro functional assay of Lp2-CAR-T cells

The calcein-based non-radioisotope cytotoxic assay demonstrated that
Lp2-CAR-T cells signiﬁcantly lysed PDPN-positive LN229/human
PDPN (hPDPN) cells but not PDPN-negative LN229 cells and Met5A cells. Lp2-CAR-T cells showed signiﬁcant dose-dependent cytolysis(effector/target ratio >6) in LN229/hPDPN cells, whereas no signiﬁcantdifference in cytolysis was observed in PDPN-negative LN229 cells with
Lp2-CAR-T and PBMCs (Figure 2A). Mock CAR-T cells were CAR-T
cells without a scFv portion. We found that mock CAR-T cells did notshow any speciﬁc cytolysis of hPDPN-transfected LN229 cells.
Interferon (IFN)-g production was measured after the co-culture ofdifferent cell lines with Lp2-CAR-T cells at an effector/target ratioof 20 for 24 h (Figure 2B). IFN-g release in the supernatant of
LN229/hPDPN cells co-cultured with Lp2-CAR-T cells produced
100.1 pg/mL of IFN-g, which was more than 10-fold higher thanthat with mock CAR-T cells (9.6 pg/mL; p < 0.05). IFN-g release after
the co-culture of PDPN-negative LN229 cells with both Lp2-CAR-T
and mock CAR-T cells was 10.9 and 12.5 pg/mL, respectively. Co-culture of TGS01 cells with Lp2-CAR-T produced 95.5 pg/mL IFN-g,
whereas mock CAR-T cells released only 7.8 pg/mL IFN-g. The production of IFN-g under the co-culture of MeT-5A cells with Lp2CAR-T and mock CAR-T was undetectable. There was no differencein the on-target effect between mock CAR-T cells and PBMCs, and
PBMCs were used as a control effector in the following experiments.
Real-time cell analysis showed that the percentages of lysis of the cancer-type PDPN-expressing glioma cells LN229/hPDPN and LN319
were signiﬁcantly higher with Lp2-CAR-T cells than with PBMCs.
This cytolytic effect was obvious after 3 h of co-culture and wasobserved continuously for 15 h (Figure 2C). However, Lp2-CAR-T
cells did not show any speciﬁc cytolysis of LN229 (GBM cells that
do not express PDPN). We also observed the cytolytic effect of
Lp2-CAR-T cells on patient-derived glioma stem cells, GIC0222
and TGS01, both of which express cancer-type PDPN. Lp2-CAR-T
cells lysed both GIC0222 and TGS01 cells (Figure 2C).
Efficacy of Lp2-CAR-T cells in combination with oncolytic virus
G47D in vitro

The co-culture of PDPN-transfected LN229/hPDPN cells and Lp2CAR-T cells produced 100.1 pg/mL IFN-g, whereas PBMCs

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Figure 1. Expression of cancer type podoplanin inglioblastoma, and construction of Lp2-chimericantigen receptor-T cells(A) Expression of cancer type podoplanin (PDPN) in glioblastoma (GBM) cell lines. Immunostaining (top, immunofluorescence; bottom, flow cytometry) using cancerspecific anti-PDPN LpMab-2 (Lp2) against PDPN-positivecancer cells (LN229/hPDPN), PDPN-negative cancer
cells (LN229), and PDPN-positive normal cells(HEK293T). LN319 and two patient-derived glioma stem
cell lines established at our institutions (GIC0222 and
TGS01) showed cancer-type PDPN expression as detected by Lp-2. Immunofluorescence for PDPN (green)is consistent with the results of flow cytometry. (B) Construction of Lp2-chimeric antigen receptor (CAR)-T cells.
Lentiviral vector construct with the EF1a promoter followed by the leader sequence, Lp-2-based single-chain
variable fragments (scFvs), CD28, 4-1BB, and CD3z.
The transduction efficiency, examined by flow cytometry
with mouse-derived F(ab’)2 recognizing biotin antibody,was 44.2% and reproducible (see also Table 1).

of Lp2-CAR-T and G47D was more effective
than Lp2-CAR-T alone in vitro. Moreover,
the antitumor effect was observed earlier
when Lp2-CAR T and G47D were used in a
combination therapy compared with anyother monotherapy.
Efficacy of Lp2-CAR-T cells on GBM in themouse subcutaneous xenograft

released 9.6 pg/mL IFN-g. When G47D, at a multiplicity of infection (MOI) of 0.1, was added to Lp2-CAR-T cells, IFN-g production increased to 152.9 pg/mL. IFN-g was not increased when
G47D was added to cultures of Lp2-CAR T and PDPN-negative
LN229 cells. The addition of G47D increased IFN-g production
from 67.1 to 98.3 pg/mL in the co-culture of TGS01 and Lp2CAR-T cells. An increase in IFN-g release was not observed
when G47D was added to PBMCs in LN229/hPDPN, LN229 and
TGS01 cells (Figure 3A). A real-time cell proliferation assay using
iCELLigence showed that either Lp2-CAR-T cells or G47D alone
suppressed cell proliferation signiﬁcantly, and the combination
further decreased cell viability in both LN229/hPDPN and
TGS01 (Figure 3B). These results showed that the combination

Subcutaneous tumors were established by injecting the mice with PDPN-transfected
LN229/hPDPN glioma cells, and treatment
was started when the mean tumor diameterreached 5 mm. G47D was injected into the tumor, and CAR-T cells were injected intravenously via the retroorbital vein. The treatment
design is summarized in Figure 4A. BothG47D and Lp2-CAR-T inhibited tumor growthsigniﬁcantly compared with the control(PBMCs and phosphate-buffered saline [PBS]). However, combination therapy signiﬁcantly inhibited tumor growth (Figure 4B). The
mean tumor volume of all four groups at day 28 of treatment showedthat mice treated with mock CAR-T cells and mock virus had significantly larger tumor size compared with both Lp2-CAR-T cell therapy
and G47D monotherapy (p < 0.05) and combination therapy(p < 0.005). Moreover, the combination therapy signiﬁcantly inhibited tumor growth compared with monotherapy (p < 0.05).
The survival curves of the four groups are shown in Figure 4C. A log
rank test indicated that while G47D- and Lp2-CAR-T-treated micesurvived longer than control mice, the combination therapy prolonged survival signiﬁcantly, and half the mice survived longer, for

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Table 1. Transduction efficiency of Lp2-CAR T
Lp2-CAR-T (batch number)

Transduction efﬁciency (%)

1

68.7

2

44.2

3

40.6

4

41

5

65.9

Median44.2

CasMab-based CAR-T therapies may improve the safety and speciﬁcity to target tumors, while the expression of cancer-speciﬁc antigen in the tumor and normal tissues should be elaborately tested in
the next preclinical trials.

We constructed a novel CasMab-based CAR T cells that targets thecancer-speciﬁc PDPN in GBM cells in this study. We successfullytransduced cancer-speciﬁc CAR on human T cells by lentivirus-mediated expression and demonstrated that the engineered T cells were
speciﬁc and effective against cancer-speciﬁc PDPN-positive cells.
These CAR-T cells showed speciﬁc cytolysis against GBM cells
in vitro, and systemic injection of these CAR-T cells signiﬁcantly inhibited the tumor growth in vivo (p < 0.05). Several CAR-T cells havebeen developed to target GBM, and a few, including epidermal growth
factor receptor (EGFR)-VIII, human epidermal growth factor receptor 2 (HER2), and interleukin (IL)-13Ra2-targeting CAR-T cells,
have been applied in clinical trials with promising results in a limited
number of patients.29-33 These mixed results point toward the factthat while CAR-T cell therapy is indeed effective against brain tumors, it does have its own limitations. Overcoming these limitations
related to the safety and efﬁcacy of CAR-T cells holds the key toimproving the therapy against brain tumors.

Although Lp2-CAR-T monotherapy was enough to moderately
inhibit tumor growth, addition of the oncolytic virus G47D to
CAR-T cell therapy drastically improved the antitumor efﬁcacy. Oncolytic viruses not only cause direct tumor killing but may also
initiate antitumor immune reactions, making the tumor microenvironment favorable for CAR-T inﬁltration. Intriguingly, the combination of G47D with Lp2-CAR-T cells elicited greater release of
IFN-g and had a greater killing effect on PDPN-expressing GBM
cells even though the combination of G47D and non-speciﬁc
T cells did not. G47D may bolster the on-target effect of speciﬁc
T cells. Several attempts have been made to enhance the efﬁcacy
of CAR-T cells against solid tumors by adding an oncolytic virus
to therapeutic strategies.37-39 Oncolytic viruses have also been
armed with chemokines40 and immune checkpoint programmed
cell death-ligand 1 (PD-L1) mini antibodies to enhance the efﬁcacy
of CAR-T cells against solid tumors.41 GBM is a heterogeneous tumor that is difﬁcult to treat by targeting a single antigen, and antigen loss after the administration of CAR-T cells is also a major
concern.42 G47D targets tumor cells independent of PDPN expression, thus killing tumor cells that evade Lp2-CAR T cells. Increasing
the antitumor efﬁcacy of CAR-T cells using another antitumor therapy may also contribute to the increased safety of CAR-T cells.
While a patient treated with a substantially high dose of HER2/
neu-speciﬁc CAR-T cells (1  1010 cells) resulted in fatality,10 subsequent studies utilizing a different HER2/neu-speciﬁc CAR at asigniﬁcantly lower dose have proven to be safe.11 Therefore, a combination therapy of CAR-T cells with an oncolytic virus may besafer than any other CAR-T cell monotherapy at a higher dose.
While we observed signiﬁcant efﬁcacy against LN229/hPDPN, an
artiﬁcial GBM cell line expressing hPDPN, the efﬁcacy against thepatient-derived tumor cells, i.e., TGS01 cells, lasted only for a shortperiod of time in the animal experiments. CAR-T cells were injected
intravenously, and G47D was administered twice intratumorally.
The dose and time schedule should be optimized in future preclinical studies.

We have previously reported the construction and efﬁcacy of NZ-1CAR-T cells that target PDPN;12 however, NZ-1 CAR-T cells may
have an on-target-off-tumor effect because they may recognize
low levels of PDPN even in normal tissues. The on-target-off-tumor
effect is a well-known adverse event of CAR-T cell therapy and canhave devastating effects on patients, even death.10,34-36 Lp2 recognizes cancer-type PDPN, which is extraordinarily glycosylated.21
Lp2 can react with PDPN-expressing cancer cells but not withnormal cells, such as mesothelium. Therefore, we produced a new
CAR using Lp-2 to overcome the on-target-off-tumor phenomenon.
We demonstrated that this novel CAR-T cell was effective against
glioma cells that express PDPN but did not have any cytolytic effectagainst PDPN-expressing non-cancer cells like mesothelial cells.

No adverse effects were observed in mice in the animal experiments.
However, it should be noted that this CAR is speciﬁc to hPDPN,
and animal experiments cannot completely simulate its effect on human tissue. Moreover, it should also be noted that this is a limitation of animal experiments in general, and thorough safety precautions should be taken before any clinical application. Although anumber of issues, such as the lack of complete response in mostof the mouse models and the clariﬁcation of the exact immunological mechanisms involved in the combination therapy, still need tobe addressed, we have successfully established novel cancer-speciﬁc
CAR-T cells against a promising tumor antigen and have reported
that it can be used in combination with an oncolytic virus against
GBM.

more than 60 days. The median overall survival (OS) durations of thefour groups were as follows: mock CAR-T (33 days), G47D (35 days),
Lp2-CAR-T (42 days), and combination of G47D and Lp2-CAR-T(51 days) (p = 0.002). In all mice, the cause of death was tumor
burden, and no side effects of CAR-T treatment were observed in
the long-term survivors in the combination group.
Against TGS01 subcutaneous tumors, the combination of Lp2-CART cells and G47D slowed tumor growth for a short period, and it did
not improve OS (Figure S1).

DISCUSSION

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Figure 2. Functional assay of Lp-2-CAR-T cells in vitro(A) PDPN-positive glioma cells (LN229/hPDPN) are lysed significantly by Lp2-CAR–T compared with peripheral blood mononuclear cells (PBMCs) and mock vector-transduced T cells (mock CAR-T) in an effector:target (E:T) ratio-dependent manner; however, specific lysis was not observed in PDPN-deficient glioma cells. Mock versus Lp2CAR-T, p < 0.05 for ET ratio of 12 or more. Means and standard deviation (SD) of 3 wells of the same experiment are shown. (B) Interferon (IFN)-g release (enzyme-linked
immunosorbent assay [ELISA]). The co-culture of LN229/hPDPN or TGS01 with Lp2-CAR-T produced approximately 90–100 pg/mL IFN-g. The IFN-g levels were significantly higher than those from mock CAR-T cells (p < 0.05). LN229 and MeT-5A cells produced only a similar amount of IFN-g when co-cultured with both Lp2-CAR-T
and mock CAR-T cells. Means and SD are shown. (C) PDPN-expressing glioma cells (LN229/hPDPN and LN319) showed significantly higher cytolysis by Lp2-CAR-T
when compared with PBMCs added at the same E:T ratio (6); *p < 0.05, for each hour starting at 4 h after addition of CAR-T cells. However, LN229 cells (which do not
express PDPN) did not show any specific cytolysis by Lp2-CAR-T when compared with PBMCs. Lp2-CAR-T cells also showed significant cytolysis in patient-derived glioma
stem cells, GIC0222 (*p < 0.05 after 5 h) and TGS01 (*p < 0.05 after 4 h). Both stem cells express PDPN. The means and standard error of the mean (SEM) of three wells of thesame experiment are shown.

This is the ﬁrst study to report a CasMab-based CAR-T cell and demonstrate the efﬁcacy of a combination therapy using CAR-T cells and theoncolytic virus G47D against GBM. Since G47D has been approved as
the ﬁrst oncolytic virus drug for malignant glioma in the world, thecombination therapy with G47D and CAR-T cells may be an effective
treatment approach for GBM. Despite its limitations, this study provides new insights into a combination therapy of CAR-T cells and oncolytic viruses to treat solid tumors, especially malignant brain tumorsthat do not respond to the current standard therapies.

MATERIALS AND METHODS
Cell lines

The human GBM cell lines (LN229, LN229/hPDPN, LN319) weremaintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM;

Sigma-Aldrich, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientiﬁc, Waltham,
MA, USA) at 37 C in a humidiﬁed atmosphere of 5% carbon dioxide (CO2). LN319 cells were purchased from Addexbio Technologies (San Diego, CA, USA). LN229 cells were purchased from theAmerican Type Culture Collection (ATCC, Manassas, VA, USA)and transfected with hPDPN to generate LN229/hPDPN cells.
Normal mesothelial cells, MeT-5A cells, were also purchased from
ATCC. The expression of hPDPN and cancer-speciﬁc PDPN on
LN229/hPDPN and Met-5A was previously described.21 The celllines were authenticated by letters when they were provided. The
GBM-initiating cells, GIC0222, were cultured in a neurobasal medium (Thermo Fisher Scientiﬁc) supplemented with 2 mmol/L
L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), N-2 and B-27

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Figure 3. Addition of the oncolytic virus G47D to aculture of Lp2-CAR-T cells(A) The combination against PDPN-expressing glioma
cells increased IFN-g release. When G47D, at a multiplicity of infection (MOI) of 0.1, was added to Lp2-CAR-T
cells, IFN-g production in the co-culture of PDPN-transfected LN229 cells (LN229/hPDPN) or TGS01 cells wasincreased significantly. There was no difference in
LN229 cells. IFN-g was not increased when G47D wasadded to PBMCs. Means and SD are shown. *p < 0.05.
(B) Lp2-CAR-T cells in combination with G47D inhibit tumor growth in vitro. A real-time cell proliferation assay
showed a gradual decrease in LN229/hPDPN cells and
TGS01 when co-cultured with Lp2-CAR-T cells. Additionof G47D to the co-culture further decreased the cell
viability. Means and SD are shown. *p < 0.05, **p < 0.01
compared with PBMCs.

phate buffer solution (PFA; FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan) for
15 min.
Blocking was performed in PBS containing 0.1%
Triton X-100 (PBST; Sigma-Aldrich) with 1.5%
goat serum for 1 h at room temperature withshaking. The coverslips were incubated withthe primary antibody Lp2, diluted to 1 mg/mL
in blocking solution for 1 h at room temperature
with shaking, followed by three washes with
PBS. The coverslips were stained using a secondary antibody, Alexa
Fluor 488 Goat Anti-Rat IgG (H5L; Thermo Fisher Scientiﬁc), and40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution(Dojindo, Kumamoto, Japan) at 1:200 dilution in the blocking solution for 30 min in the dark, followed by three washes with PBST.
They were then mounted onto slides. Stained cells were observed under a ﬂuorescence microscope (Keyence BZ-X710, Osaka, Japan), andpictures were taken.

supplements (Thermo Fisher Scientiﬁc), recombinant human basicﬁbroblast growth factor (bFGF) and EGF (16.7 ng/mL each; R&D
Systems, Minneapolis, MN, USA), 100 units/mL penicillin, and100 mg/mL streptomycin (Thermo Fisher Scientiﬁc) at 37 C in ahumidiﬁed atmosphere of 5% CO2. Glioma-derived cancer stem
cells, TGS01, were cultured in DMEM/F12 medium (Thermo Fisher
Scientiﬁc) supplemented with 200 mM L-glutamine (Sigma-Aldrich), 45% D-glucose solution (Sigma-Aldrich), B-27 supplements(GIBCO, Thermo Fisher Scientiﬁc), recombinant human bFGF, and
EGF (20 ng/mL each; Peprotech). GIC0222 and TGS01 were primary cultured GBM cells from the GBM tissue sample taken after
obtaining written informed consent from all patients undergoing
surgery at the Nagoya University Hospital and the Tokyo University
Hospital, Japan, respectively. This study was approved by the two
institutional review boards. The dissociation procedures have been
described elsewhere. All cell lines were tested for the presence of
mycoplasma contamination.

The mAb Lp2 has been previously established.21 The scFv was thenproduced, and the scFv portion in the previous CAR construct,
pELNS-empty-CAR,43 was changed to the Lp2-based scFv to generatepELNSLp2-CAR by gene synthesis (Genscript Japan, Tokyo,
Japan).12 In this construct, the EF1a promoter drives the CAR fusion
protein containing the Lp2-based scFv targeting PDPN, CD28,
4-1BB, and CD3z domains.

PDPN immunofluorescent staining

Preparation of Lp2-CAR T and mock CAR T cells

GBM, GIC0222, and TGS01 cell lines were plated on a 24-well platecontaining BD BioCoat poly-L-lysine cellware 12-mm round coverslips with 10% FBS/DMEM and incubated for 24 h.

Lp2 (cancer-speciﬁc anti-PDPN)-CAR-T and mock (no scFV)CAR-T cells43 were prepared by the production and transduction oflentiviral vectors. HEK293T cells (8  106) were plated in 175-cm2
ﬂasks at 37 C in a humidiﬁed atmosphere of 5% CO2. At 24 h, theSIN vector, pMDLg/pRRE, pRSV-Rev, and pMD2.G were co-transfected using the X-tremeGENE 9 DNA transfection reagent (Roche

The coverslips were rinsed twice with phosphate-buffered saline(Thermo Fisher Scientiﬁc) and placed in 4% paraformaldehyde phos-

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Construction of a self-inactivating (SIN) lentiviral vector

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Figure 4. Efficacy of Lp2-CAR-T cells in combination
with the oncolytic virus G47D in vivo(A) Experimental schema, subcutaneous tumor was established by injecting 6-week-old NOD/Shi-scid, IL2Rr KO Jic(NOG) mice with LN229/hPDPN glioma cells. Treatment
was started when tumor diameter reached 5 mm. G47D
was injected into the tumor, and CAR-T was injected intravenously via the retroorbital vein. On day 4, G47D intratumoral injection was repeated. i.v., intravenous injection;OS, overall survival. (B) Tumor volumes determined bycaliper measurements. Means and SEM are shown (n = 6
or 7, respectively). Representative of the two experiments.
*p < 0.05, **p < 0.005 by two-way analysis of variance(ANOVA). (C) Median overall survival (OS) durations of thefour groups (n = 6 or 7) were 33, 35, 42, and 51 days,
respectively. p = 0.002 by log rank test. While G47D- andLp2CAR-treated mice survived longer than the control
mice, the combination therapy prolonged their survival
significantly, and half the mice survived long term.

5% CO2 for 24 h. Next, the medium (3 mL/well)
was gently removed without disturbing the clustering of PBMCs, and the lentiviral pELNS-Lp2CAR vector supernatant (X10; 3 mL/well) was
added, and the cells were cultured for 24 h. To
produce mock CAR-T cells, 3 mL medium wasremoved in a similar fashion, and 3 mL fresh medium was added to each well, followed byculturing of the cells for 24 h. The medium wasthen replaced with fresh medium, and the cells
were cultured for 48 h. The same batch of
PBMCs was used to prepare both mock CAR-T
and Lp2-CAR-T cells for each experiment.
Flow cytometric analysis of Lp2-CAR
expression

Applied Science, Penzberg, Germany). The supernatant was collected
at 48 h, mixed with PEG-it Virus Precipitation Solution (SBI Funakoshi, Tokyo, Japan), and incubated for 24 h at 4 C. The supernatant/PEG-it mixture was then centrifuged at 1,500  g for 30 min
at 4 C. The pellet was resuspended in 1/10 of the original volume using a cold sterile medium at 4 C and was stored at 80 C.
Freshly harvested heparinized PBMCs from healthy donors wereseparated over a monolayer of Ficoll-Paque PLUS (1,000  g for
20 min at 20 C; GE Healthcare Bio-sciences AB, Uppsala, Sweden).
PBMCs were cultured in 4 mL AIM-V medium (Thermo Fisher Scientiﬁc) with 2.5% human serum per well of a 6-well plate coated with
anti-human CD3 mAbs (Thermo Fisher Scientiﬁc) in the presence of
IL-2 (50 units/mL; PeproTech) at 37 C in a humidiﬁed atmosphere of

As the construct contained murine-derived fragment antigen-binding region (Fab), Lp2-CAR
expression on the cell surface of PBMCs was
examined by ﬂow cytometry using either
FACSCanto II equipped with the CellQuest Pro software (BD Biosciences, Franklin Lakes, NJ, USA) or Cytoﬂex (Beckman Coulter, Tokyo, Japan). PBMCs were washed twice with PBS containing 0.5%
bovine serum albumin (BSA; Sigma-Aldrich) and 2 mmol/L ethylenediaminetetraacetic acid (EDTA) (Dojindo). PBMCs were then incubated with biotin-AfﬁniPure F(ab’)2 fragment-speciﬁc goat antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove,
PA, USA) at 4 C for 30 min.
After washing twice, PBMCs were stained with streptavidin (SA)phycoerythrin (PE; BD Biosciences) at 4 C for 30 min in the dark.
After washing twice, PBMCs were suspended in 1% PFA and analyzed
using ﬂow cytometry Cytoﬂex. Data were analyzed using FlowJo v.10
(BD Biosciences, Franklin Lakes, NJ, USA).

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Analysis of IFN-g production by effector cells

Subcutaneous glioma xenograft modelIFN-g production by effector cells was measured by enzyme-linked
immunosorbent assay (ELISA) using the human IFN-g ELISA
Ready-SET-Go! (Thermo Fisher Scientiﬁc). Effector cells (1 106
unsorted Lp2-CAR-transduced PBMCs or non-transduced
PBMCs) were co-cultured with 1104 target cells (LN229, LN229/
hPDPN, TGS01, or MeT-5A) in each well of a 96-well plate for
24 h. The cell culture supernatants were harvested and used for
ELISA. Cancer-type PDPN was highly expressed in LN229/
hPDPN cells but not in LN229cells. Therefore, these cells wereused as the positive and negative controls, respectively. While
PDPN is also expressed in MeT-5A cells, it is not a cancer cell;
therefore, the non-reactivity of MeT-5A cells to Lp2-CAR-T cells
would serve as a conﬁrmation of cancer speciﬁcity of Lp2-CAR-T
cells.

Immunoﬂuorescence analysis indicated that PDPN was highly expressed in LN319 cells. However, this cell line is not tumorigenic
in vivo.44 Thus, we utilized LN319 cells only in the in vitro experiments. LN229/hPDPN cells were found to be tumorigenic in preliminary experiments. Therefore, LN229/hPDPN cells were used inanimal experiments. All animal experiments were approved by the
ethics committee of The University of Tokyo and were performedin accordance with our institutional animal care and use guidelines.
Six-week-old NOD/Shi-scid, IL2Rg null (NOG) female mice (Central
Institute for Experimental Animals, Kawasaki, Japan) were used forthe experiments.

Cytotoxicity assay

Target cells (LN229, LN229/hPDPN, LN319, Met-5A, GIC0222, and
TGS01) were suspended at a ﬁnal concentration of 1  106 cells/mL
and incubated with 10 mmol/L (100 times) calcein-AM solution(Dojindo) at 37 C for 30 min with occasional shaking. Afterwashing twice, the cells were adjusted to 1  105 cells/mL, and1  104 cells (100 mL) were placed into a well of a round-bottom
96-well plate. Effector cells (NZ-1-CAR-transduced PBMCs, 3C10CAR-transduced PBMCs, or non-transduced PBMCs) were incubated for 48 h. The cells were then harvested and added to eachwell at an appropriate effector:target ratio of 50:1, 25:1, 12.5:1, 6:1,
and 3:1 and incubated at 37 C for 5 h. After centrifugation at
300  g for 2 min, 75 mL supernatant was aspirated carefully
and loaded into a 96-well white/clear ﬂat-bottom plate (BD Biosciences). The absorbance was then measured to evaluate the tumorkilling efﬁcacy.

Mice were anesthetized intraperitoneally (i.p.) with a solution of ketamine (75 mg/kg body weight) and xylazine (100 mg/kg bodyweight). After shaving the left ﬂank of the mice, 1  106 LN229/
hPDPN cells/mouse suspended in 100 mL PBS were injected subcutaneously. When the mean tumor diameter reached 5 mm, mice wererandomized and treated with an intratumoral injection of either
PBS or 4  104 PFU of G47D, followed by intravenous injection ofeither Lp2-CAR-transduced PBMCs or non-transduced PBMCs.
PBMCs (2  106 cells) were suspended in 200 mL PBS and infused
intravenously (i.v.) via the retroorbital vein. Untreated mice were
infused with PBS alone. An intratumor injection with either PBS or
4  104 PFU of G47D was repeated once, 4 days after the ﬁrst treatment; however, no additional CAR-T cells were administered. Tumor
size and survival were monitored following tumor inoculation. Tumor dimensions were measured with a digital caliper, and tumor volumes were calculated as follows: volume = (length  width  height)/
2. The mice were euthanized when the tumor length reached 20 mm
or when the tumor volume reached 1,600 mm3.
Statistics

Target cells in the medium with 3 mL of 10% sodium dodecyl sulfate(SDS; Wako, Osaka, Japan) were used to determine the maximum
release, and target cells alone were used to measure the spontaneous
release. The percentage of speciﬁc lysis was calculated as follows:
100  (experimental releasespontaneous release)/(maximum releasespontaneous release).

Student’s t test was used to determine the statistical signiﬁcance of differences between the two groups, and a two-tailed p value of <0.05
was considered to be statistically signiﬁcant. Mouse survival curves
were obtained using the Kaplan-Meier method, and the difference between curves was compared using the log rank test with Bonferroni’s
correction.

Cytotoxicity assay using real-time cell analyzer (RTCA)

DATA AND CODE AVAILABILITY

Kinetic analysis of tumor cell lysis was performed using iCELLigenceand xCELLigence RTCA (ACEA Biosciences, San Diego, CA, USA).
Twenty thousand tumor cells were seeded onto E-Plate L8 PET(ACEA Biosciences). After 24-h culture, T cells or control media
were added. The cell index (CI) was recorded every 1 h, and cytotoxicity was calculated as {CI (control medium)CI (effector cells)}/CI(control medium).

Experimental data and applicable methodology documents may beavailable upon request to the corresponding authors (A.N. and T.T.).

SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.
1016/j.omto.2022.07.006.

ACKNOWLEDGMENTS
To analyze the additional effect of adding G47D, the tumor cells wereﬁrst infected with G47D after 24 h of culture, and the supernatant
was replaced with fresh culture media after 1 h of infection. After
another 1 h culture, T cells or control media were added, and CI was recorded every hour.

272

Molecular Therapy: Oncolytics Vol. 26 September 15 2022

T.T. owns the patent for G47D in Japan. This work was supported inpart by a Grant-in-Aid for Young Scientists (L.C., 19K18416); theFund for the Promotion of Joint International Research B (A.N.,
19KK0228); a Grant-in-Aid for Scientiﬁc Research on Innovative
Areas (A.N., 17H06356); a Grant-in-Aid for Scientiﬁc Research B

www.moleculartherapy.org(A.N., 20H03789); the Practical Research for Innovative Cancer Control, Japan Agency for Medical Research and Development (T.T.,
AMED; JP18ck0106416), the Translational Research Program,
AMED (T.T., JP20lm0203140); the Science and Technology Platform
Program for Advanced Biological Medicine, AMED (Y.K.,
JP21am0401013); and BINDS, AMED (Y.K., JP21am0101078). We
would like to thank Ms. Junna Natsume for her editorial assistance.

AUTHOR CONTRIBUTIONS
Study concept and design, A.N. and T.T.; provision of study materials,
Y.K.; data collection and assembly, L.C., A.K., M.O., S.M., A.Y., S.K.,
S.S., S.O., and J.Y.; data analysis and interpretation, A.N. and T.T.;
development of tables and ﬁgures, L.C., H.T., Y.R., and A.N.; writing
the report, L.C., A.N., and T.T.; critical review of the manuscript andapproval of the ﬁnal version, all authors.

DECLARATION OF INTERESTS
The authors have no conﬂict of interest to declare.

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