Received: 4 February 2022

Revised: 20 June 2022

Accepted: 22 June 2022

DOI: 10.1111/gtc.12972

ORIGINAL ARTICLE

Improved anti-solid tumor response by humanizedanti-podoplanin chimeric antigen receptor transduced
human cytotoxic T cells in an animal modelAkihiro Ishikawa1
Mika K. Kaneko2

Masazumi Waseda1| Tomoko Ishii1| Yukinari Kato2,3| Shin Kaneko1||

1

Shin Kaneko Laboratory, Department of
Cell Growth and Differentiation, Center
for iPS Cell Research and Application(CiRA), Kyoto University, Kyoto, Japan

Abstract
Recently, research has been conducted with chimeric antigen receptor (CAR)-T
cells to improve efficacy against solid tumors. Humanized CAR improved the long-2

Department of Antibody Drug
Development, Tohoku University
Graduate School of Medicine, Sendai,
Japan

3

Department of Molecular Pharmacology,
Tohoku University Graduate School of
Medicine, Sendai, Japanterm survival of CAR-T cells in patients' peripheral blood, resulting in increased
therapeutic efficacy. Therefore, the humanization of the CAR-gene sequence isconsidered an effective method. Podoplanin (PDPN) is a glycosylated transmembrane protein that is highly expressed in solid tumors and is associated with poor
prognosis in patients with cancer. Therefore, PDPN is considered a biomarker andgood target for cancer treatment with CAR-T cells. Previously, an anti-PDPN CAR

Correspondence
Shin Kaneko, Shin Kaneko Laboratory,
Department of Cell Growth andDifferentiation, Center for iPS Cell
Research and Application (CiRA), Kyoto
University, Kyoto, Japan.
Email: kaneko.shin@cira.kyoto-u.ac.jp
Funding information
Japan Agency for Medical Research andDevelopment, Grant/Award Numbers:
JP21am0101078, JP21am0401013,
JP21bm1004001, JP22ama121008; iPS Cell
Research Fundwas generated from a conventional nonhumanized antibody—NZ-1, the only antiPDPN antibody for which a CAR was produced. In this study, we investigatedother anti-PDPN CARs from the antibody NZ-27, or humanized NZ-1, to enhance
the therapeutic potential of CAR-T cells. The CAR signal intensity was enhancedby the efficient expression of CAR proteins on the T-cell surface of NZ-27 CAR-T
cells, which show tumor-specific cytotoxicity, proinflammatory cytokine production, and anti-tumor activity against PDPN-expressing tumor xenografts in micethat were significantly better than those in nonhumanized NZ-1 CAR-T cells.
KEYWORDS

CAR-T cells, chimeric antigen receptor, humanized antibody, podoplanin, solid tumor

Communicated by: Yoshimi Takai

1 | INTRODUCTION
A chimeric antigen receptor (CAR) is an artificially generated receptor with a monoclonal antibody-derived singlechain variable fragment (scFv) in its extracellular domain
and CD3z or co-stimulatory molecules in its intracellulardomain (June et al., 2018; Scarfò & Maus, 2017). CAR-T
Akihiro Ishikawa and Masazumi Waseda contributed equally tothis work.

cells targeting CD19-positive B-cell malignancies were thefirst therapeutic CAR-T cell products with excellent therapeutic efficacy in prolonging patient survival. Recently,
better therapeutic efficacy was reported to be achieved by
the prolonged persistence of CAR-T cells in patients. One
strategy to prolong CAR-T endurance involves the humanization of the animal-derived scFv sequences of CAR.
Additionally, clinical trials have reported higher therapeutic efficacy using humanized CD19 CAR-T cells thanoriginal CD19 CAR-T cells (Myers et al., 2021).

© 2022 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.
Genes Cells. 2022;27:549–558.

wileyonlinelibrary.com/journal/gtc

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ISHIKAWA ET AL.

LN319, CHO/hPDPN, and MG-63 cells expressed PDPN
(Figure 1a), and we confirmed that NZ-1 and NZ-27 scFvFc
were able to specifically and similarly bind to PDPNexpressing cells (Figure 1b).

Podoplanin (PDPN) is a mucin-type transmembraneprotein with a glycosylated extracellular domain that is overexpressed in various cancers, such as brain, oral, and lungcancers (Breiteneder-Geleff et al., 1997; Chu et al., 2005;
Kahn et al., 2002; Kato et al., 2003, 2004; Mishima
et al., 2006; Schacht et al., 2005), and is considered
a promising therapeutic target for cancer treatment (Kreppel
et al., 2010; Mori et al., 2021; Waseda & Kaneko, 2020). To
target PDPN, we previously focused on the rat anti-human
PDPN antibody (NZ-1), which recognizes PDPN specifically(Kato et al., 2006). The therapeutic efficacy of NZ-1-scFvimmunotoxin and NZ-1-derived CAR-T cells has beenreported in PDPN xenograft tumor-engrafted mousemodels (Chandramohan et al., 2013; Shiina et al., 2016).
The effectiveness of CAR-T cells partially depends on
CAR expression on the T cell surface and CAR signal
intensity (Jayaraman et al., 2020). In this study, toincrease the therapeutic potential of anti-PDPN CAR-T
cells, we generated a CAR from the NZ-27 antibody,which is a humanized NZ-1 antibody. This study clarified
the improvement of CAR signal intensity by efficiently
expressing CAR proteins on the T-cell surface andreported that the therapeutic efficacy of NZ-27 CAR-T
cells was better than that of NZ-1 CAR-T cells against
PDPN-expressing cells in vitro and in vivo.

2.2 | NZ-27 CAR-T cells enhanced theircytotoxicity and proinflammatory cytokine
production against PDPN-expressing cells
compared to NZ-1 CAR-T cells
Third-generation CARs were generated from the scFv
part of NZ-1 and NZ-27, and retroviral vectors containing
the CARs were transduced into primary T cells(Figure 2a). The transduction efficiency of each CAR
gene was evaluated using TagBFP2 fluorescence. Among
NZ-1 and NZ-27 CAR-T cells, 13.2 and 13.0% were CD4and TagBFP2-positive cells and 59.7 and 66.2% were CD8and TagBFP2-positive cells; totally, 78.6% and 83.5%
TagBFP2-positive cells were successfully obtained from
NZ-1 and NZ-27 CAR-T cells, respectively (Figure 2b).
This result suggests that (1) there was no significant
difference in transfection efficiency between NZ-1 and
NZ-27 and that (2) NZ-1 and NZ-27 were efficiently
transduced into both CD4 and CD8 T cells.
To evaluate whether NZ-1 and NZ-27 CAR-T cells
were effective against PDPN-expressing cells, we performed a cytotoxicity assay of CAR-T cells against
PDPN-expressing cells. Each CAR-T cell showed E:T
ratio-dependent
cytotoxicity
against
LN319,
CHO/hPDPN, and MG-63 cells, which express PDPN
(Figure 2c). NZ-27 CAR-T cells showed significant cytotoxicity against LN319 and CHO/hPDPN cells comparedto that against NZ-1 CAR-T cells, but not against MG-63

2 | R E SUL T S
2.1 | NZ-1 and NZ-27 scFvFc antibodies
bind to PDPN-expressing cells
To evaluate whether NZ-1 and NZ-27 scFvFc are effectively
attached to PDPN, we first selected PDPN-expressing cells.

(a)

% Max

CHO-K1

LN319

CHO/hPDPN

MG-63

PDPN-KO LN319

100

100

100

100

100

80

80

80

80

80

60

60

60

60

60

40

40

40

40

40

20

20

20

20

20

0

0

0

0

3
10

0

4
10

5
10

3
10

0

4
10

5
10

3
10

0

4
10

5
10

Unstained control
Podoplanin IgG(clone : NC-08)

0

3
10

0

4
10

5
10

3
10

0

4
10

5
10

PDPN

(b)
CHO-K1

CHO/hPDPN

LN319

PDPN-KO LN319

MG-63

Isotype control
NZ-1 scFvFc
NZ-27 scFvFc
0

2
10

3
10

4
10

5
10

0

2
10

3
10

4
10

5
10

0

2
10

3
10

4
10

5
10

0

2
10

3
10

4
10

5
10

0

2
10

3
10

4
10

5
10

scFvFcF I G U R E 1 NZ-1 and NZ-27 scFvFc antibodies bind to PDPN-expressing cells. (a) Flow cytometry plots of PDPN expression of each cell.
(b) NZ-1 and NZ-27 scFvFc antibodies binding to PDPN-expressing cells

ISHIKAWA ET AL.

551

F I G U R E 2 NZ-27 CAR-T cells enhanced their cytotoxicity and cytokine production against PDPN-expressing cells compared to NZ-1
CAR-T cells. (a) Schematic of retroviral constructs encoding NZ-1 and NZ-27 CARs. These were expressed with the IRES-linked TagBFP2
gene. (b) Flow cytometry plots of CD4, CD8, TagBFP2-positive CAR-T cells. (c) In vitro cytotoxicity assay of each CAR-T cell measured using
nonradioactive cellular cytotoxicity assay using LN319, PDPN-KO LN319, CHO-K1, CHO/hPDPN, and MG-63 cells (n = 5). (d) IFN-γ, IL-2,
and TNF expression of each CAR-T cell against PDPN-expressing cells (n = 3). Data represent mean ± standard error of the mean ofn independent experiments. *p < .05, **p < .01, and ****p < .001 using one-way analysis of variance with Tukey's multiple comparison test.
CAR, chimeric antigen receptor; IFN-γ, interferon γ; IL-2, interleukin 2; IRES, internal ribosome entry site; PDPN, podoplanin; TNF, tumor
necrosis factor

552

ISHIKAWA ET AL.

(a)
LN319
subcutaneous injection

CAR-T cell
intraveneous injection

day0

day7

day14, 21, 28, 35, 42

200

NZ-1
T cellinjection

150

NZ-27
CAR(-)
PBS

100
50
0
0

10

20

30

40

mCD45 (-), hCD45 (+) [%]

(c)

(b)

Tumor volume [mm3]Tumor volume
mouse peripheral blood analysis
Immunohistochemical staining (day21)

NZ-1

1.5

NZ-27
CAR(-)

1.0

PBS

0.5

0.0

50

10

Days after tumor innoculation

20

Days after tumor innoculation

(d)
NZ-1

NZ-27

CAR(-)HE staining

PBS

30

F I G U R E 3 NZ-27 CAR-T cells havesuperior anti-tumor activity against the
PDPN xenograft model compared to
NZ-1 CAR-T cells. (a) Subcutaneous andintravenous injections of LN319 cells
and CAR-T cells, respectively.
(b) Monitored for tumor volume.
(c) mCD45 , hCD45+ cells in mouse
peripheral blood (three mice). (d) Antihuman CD3 antibodies were used for
immunohistochemical staining of LN319
cells derived from subcutaneous
carcinoma in mice treated using
different CAR-T cells. The dotted line
indicates human CD3+ cells. Data
represent mean ± standard error of themean of n independent experiments.
****p < .001 using one-way analysis ofvariance with Tukey's multiple
comparison test. CAR, chimeric antigen
receptor

Anti-humanCD3 (10X)

cells (Figure 2c). NZ-27 scFvFc showed lower affinity to
MG-63 cells compared to NZ-1 scFvFc (Figure 1b), suggesting that this is one of the reasons underlying the low
cytotoxicity of NZ-27 CAR-T cells against MG-63 cells.
Because T-cell cytotoxicity is known to be influenced by
the production of proinflammatory cytokines, we evaluated IFN-γ, IL-2, and TNF activation for 24 h after coculturing CAR-T cells with PDPN-expressing cells. The
results showed significantly better expression of IFN-γ,
IL-2, and TNF in the supernatant samples containing
NZ-27 CAR-T cells than in those containing NZ-1 CAR-T
cells (Figure 2d).

2.3 | NZ-27 CAR-T cells have superioranti-tumor activity against the PDPN
xenograft model compared to NZ-1
CAR-T cells
To evaluate the anti-tumor activity of different CAR-T
cells against PDPN-expressing cells, LN319 cells were
subcutaneously injected into NOG mice. Seven days after
subcutaneous tumor inoculation, other CAR-T-cells were
injected intravenously. Tumor volume and hCD45+ T
cell proportion in mouse peripheral blood and T-cell

infiltration into the tumor were evaluated (Figure 3a). As
indicated in Figure 3b, tumor progression was suppressedfor 14 days in all CAR-T cell-treated groups. The volume
recurrently increased in the NZ-1 CAR-T cell-treatedgroup, whereas NZ-27 CAR-T cell-treated groups did notshow recurrent tumor progression until day 42.
Since the long-term persistence of T-cells in peripheral
blood and the number of infiltrated T-cells in tumor correlate with therapeutic efficacy against cancer (Chen &Mellman, 2013; Park et al., 2016), hCD45+ cell populations in mouse peripheral blood from each T-cell-treatedgroup were periodically evaluated; at 14 days after T-cell
injection, tumors were immunohistochemically stainedwith anti-human CD3 antibodies. hCD45+ cell populations were maintained for 17 days after T-cell injection;
however, no significant difference was observed in
hCD45+ cell populations in each CAR-T cell-treatedgroup (Figure 3c). Next, we observed the existence of each
CAR-T cell that infiltrated the tumor. The group treatedwith NZ-27 CAR-T cells had more human CD3-positive
infiltrated cells than that treated with NZ-1 CAR-T cells(Figure 3d).
These results suggest that NZ-27 CAR-T cells
improved their proliferation against PDPN-expressing
solid tumors compared to NZ-1 CAR-T cells.

ISHIKAWA ET AL.

553

2.4 | Expression of NZ-1 CAR on the cellsurface was weakened, making T-cell
activation difficult compared toother CARs

effector function and anti-tumor efficacy against PDPNexpressing cells. This result was thought to be due to thelow expression of NZ-1 CAR on the cell surface and theinsufficient total amount of T-cell activation signaling.
The Myc tag sequence was inserted downstream of thesignal sequence of each CAR to determine the expression
level of each CAR on the cell surface, as shown in
Figure 4a. Each CAR was expressed in Jurkat NFAT-luc

We showed that NZ-1 and NZ-27 scFvFc have similar
specific affinity against PDPN. However, compared with
NZ-1 CAR-T cells, NZ-27 CAR-T cells showed better

(a)

5'LTR

NZ-1
SS

NZ-1
scFv

CD8
TM

CD28 4-1BB CD3ζ

IRES TagBFP2

3'LTR

5'LTR

NZ-27
SS

NZ-27
scFv

CD8
TM

CD28 4-1BB CD3ζ

IRES TagBFP2

3'LTR

Myc-Tag(c)(d)

CAR expression

NZ-27 peakNZ-1 peak

Relative expression(vs gapdh)

0.25

CAR(-)
NZ-1
NZ-27
5
10

MFI : 708

MFI : 2141

0.05
0.00

kDa
116
66

CAR

kDa
116

CytosolMembrane

-1
-27
NZ NZ

-1
-27
NZ NZ

66

CAR

66

(-)

27

R
A

CAR

40

40

40

kDa
116

0.3
0.2
0.1

Cytosol

C

N

1
Z-

NZ

kDa

N

1
Z-

NZ

-27

Membrane
kDa

116

116

116

66

66

66

40

GAPDH 40GAPDH 40

N

1
Z-

NZ

-27

Na, K-ATPase

(g)

0.10
0.05

0.1

0.0
NZ-1

NZ-27

0.2

NZ-27

NZ-1

NZ-27

2000

luminescence [RLU]

kDa

AR

0.15

Na, K-ATPase

GAPDH
-27

0.20

0.00
NZ-1

(-)

0.3

0.25

0.4

0.0

Total

Membrane

ns

Relative expression(vs GAPDH)

C

-1
-27
NZ NZ

5
10

Cytosol

Total

Relative expression(vs GAPDH)

-)

4
10

C

N

Total

3
10

Myc-Tag(f)

Myc-Tag
(
AR

2
10

0

Z-

TagBFP2(e)

CAR(-)
NZ-1
NZ-27

0.10

1

4
10

0.15

Z-

3
10

MFI : 684

N

2
10

0

0.20

Relative expression(vs Na,K-ATPase)

(b)

NZ-1

1500

NZ-27
1000
500
0
CHO-K1

CHO/hPDPNF I G U R E 4 Expression of NZ-1 CAR on the cell surface is weakened, making it difficult for T-cell activation compared to other CARs.
(a) Schematic of retroviral constructs encoding Myc-Tag-inserted NZ-1 and NZ-27 CARs. These CARs were expressed with the IRES-linked
TagBFP2 gene. (b) Flow cytometry plots of each Jurkat NFAT-luc cells with TagBFP2 expression. (c) Each CAR transcription activity wasevaluated using qPCR (n = 3). (d) Flow cytometry plots of each Jurkat NFAT-luc cells with Myc-Tag expression. Dotted lines indicate thepeaks of NZ-1 and NZ-27 CAR-expressing Jurkat NFAT-luc cells. (e) Western blotting assay of each Jurkat NFAT-luc cells with Myc-Tag
expression. The results showed expression of Myc-Tag, GAPDH, and Na, K-ATPase. (f) Statistical analysis of results generated by Western
blotting (n = 3). (g) Luciferase luminescence derived from each CAR stimulation (n = 3). Data represent mean ± standard error of the meanof n independent experiments. **p < .01, *p < .05, and ns using one-way analysis of variance with Tukey's multiple comparison test orStudent t-test554

cells, a cell line derived from human acute T-cell leukemia cells genetically engineered to express luciferase
under NFAT response element upon TCR signalmediated activation (Clipstone & Crabtree, 1992; Lyakh
et al., 1997). As shown in Figure 4a, retroviral vectors
were used for gene transfer into Jurkat NFAT-luc cells.
The transduction efficiency of each retroviral vector was
evaluated by TagBFP2 and mRNA expression at the samelevel (Figure 4b,c). However, the same CAR mRNA andTagBFP2 protein expression levels were confirmed.
Myc-Tag expression, which reflects cell surface CAR
expression, was weakened in NZ-1 CAR-expressing Jurkat NFAT-luc cells compared with NZ-27 CAR-T cells(Figure 4d). We further observed NZ-1 and NZ-27 CAR
total protein expression levels using the Myc tag. There
was no significant difference in the total protein expression levels of NZ-1 and NZ-27 CARs, which was the same
result as that obtained with mRNA expression (Figure 4c,e,f). Next, we observed the localization of each CAR andfound that NZ-1 CARs and NZ-27 CARs were significantly more abundant in the cytoplasm and plasma
membrane, respectively (Figure 4e,f).
Finally, to estimate the strength of TCR signals
induced by CAR activation, we co-cultured Jurkat
NFAT-luc cells expressing each CAR with CHO/hPDPN
cells and quantified luciferase luminescence. Luciferase
luminescence was elevated in the co-culture with
CHO/hPDPN cells (Figure 4g). Moreover, the luciferase
luminescence of NZ-27 CAR-expressing Jurkat NFAT-luc
cells was higher than that of NZ-1 CAR-expressing Jurkat
NFAT-luc cells (Figure 4g).
These results suggest that one of the reasons for theenhanced anti-tumor effect of NZ-27 CAR-T cells isenhanced T-cell activation associated with higher cell surface expression of NZ-27 CAR compared with that of
NZ-1 CAR.

3 | DISCUSSION
In this report, we showed that the different expression
levels of NZ-1 and NZ-27 CAR on the cell surface affectthe anti-tumor efficacy of CAR-T cells. The effector
function of the CAR-T cell depends on the efficiency of
insertion and expression of the CAR gene; however,there were no significant differences in the mRNA
expression level of NZ-1 and NZ-27 CAR and the total
protein expression level of the Myc-Tag inserted into
the CAR sequence. These results suggest that theenhanced effector function of NZ-27 CAR-T cells was
not due to differences in CAR gene insertion andexpression levels. Furthermore, NZ-27 CAR expression
on the plasma membrane was higher than that of NZ-1

ISHIKAWA ET AL.

CAR, suggesting the enhanced effector function of
NZ-27 CAR-T cells.
PDPN has four platelet aggregation-stimulating(PLAG) domains in the extracellular domain that bind to
C-type lectin-like receptor-2 (CLEC-2). CLEC-2 binds to
the PLAG3 domain of PDPN and has been reported to promote cancer cell metastasis (Waseda & Kaneko, 2020).
Therefore, the NZ-27 CARs generated in our study will
have better anti-tumor activity against PDPN-expressing
cells and may preferentially have the potential to suppress
cancer metastasis because the NZ-27 epitope is located in
the PLAG2-3 domain (Ogasawara et al., 2008).
The scFv that constitutes the CAR contains
complementarity-determining regions (CDRs) andframework (FR) regions, which are responsible for antigen specificity and stability(Fujiwara et al., 2020; Guoet al., 2022). The CDR and FR sequences in scFv differ
among species, and FR sequences have been reported
to change into human immunoglobulin sequences toaccommodate immunogenicity in the creation of humanized CAR (Guo et al., 2022). In this report, we created
NZ-27 CAR by optimizing the CDR of the NZ-1 CAR tothe human codon sequence and changed the FR
sequence to that of trastuzumab, a human IgG1 antibody.
This change did not alter the mRNA transcript levels or
total protein levels of the NZ-1 and NZ-27 CARs but
resulted in a significant difference in the expression levelsof the CARs on the plasma membrane. It has also beenreported that changing only the CDR does not change
the total amount of protein but instead changes theamount and stability of expression on the cell membrane(Fujiwara et al., 2020). After a gene is transcribed, various
post-translational modifications occur in intracellularorganelles and are thought to determine the function,
localization, and stability of the protein; thus, we speculated that the difference in post-translational modifications on each CAR and structural differences in CDR
and/or FR due to humanization may have caused theexpression difference (Moremen et al., 2012; Rape, 2018;
Yang & Qian, 2017). The possibility of post-translation
modifications related to efficient protein formation andexpression on the cell surface in human cells to create
highly effective CAR is an interesting topic of study andshould be further explored.
In this report, we performed an in vivo experimentusing only the LN319 cell line. Therefore, we cannot
exclude the possibility that the improvement of anti-tumor
efficacy on using NZ-27 CAR-T cells is applicable to the
LN319 cell line. In a further study, we would like to evaluate NZ-27 CAR using other PDPN-expressing cell linesand develop a new CAR for PDPN-expressing tumors.
In summary, herein, we demonstrated the usefulness
of NZ-27 CAR for PDPN-expressing cells. We found that

ISHIKAWA ET AL.

in addition to the strength of the affinity for the antigen,the enhancement of activation signals associated withhigher CAR expression on the T cell surface is essential
for creating functional CAR. Based on the findings of thisstudy, the clinical application of NZ-27 CARs withimproved therapeutic efficacy against PDPN-expressing
cancer cells is anticipated.

4 | EXPERIMENTAL PROCEDURES
4.1 | Ethical consideration
This study was conducted in accordance with the tenetsof the Declaration of Helsinki and was approved by theInstitutional Ethical Board of Kyoto University. The Institutional Animal Research Committee approved animal
experiments, and all experiments were performed inaccordance with relevant guidelines and regulations.
Healthy volunteers (from whom PBMCs were obtained)
provided written informed consent.

4.2 | Cell lines and peripheral bloodmononuclear cells
LN319 and CHO-K1 were purchased from Addexbio
Technologies (San Diego, CA) and ATCC (Manassas, VA,
USA), respectively. PDPN-KO LN319 and CHO/hPDPN
cells were initially generated in our previous study(Kato & Kaneko, 2014). The RIKEN BRC provided
the human osteosarcoma cell line MG-63 (RCB1890)
through the National BioResource Project of the MEXT/
AMED, Japan. LN319 and PDPN-KO LN319 cells weremaintained in Dulbecco's modified Eagle medium
supplemented with 10% fetal bovine serum (FBS) and2 mM penicillin–streptomycin-glutamine (PSG) solution(Sigma–Aldrich Corp., St. Louis, MO). CHO-K1 andCHO/hPDPN cells were maintained in RPMI-1640
medium (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan) supplemented with 10% FBS and 2 mM
PSG solution. MG-63 cells were maintained in minimum
essential media (MEM) (Thermo Fisher Scientific Inc.,
Waltham, MA) supplemented with 10% FBS and 2 mM
PSG solution. Peripheral blood mononuclear cells(PBMCs) were used to prepare the primary CAR-T cells.
PBMCs were obtained from healthy volunteers. T cells
were cultured in α-MEM (Sigma–Aldrich) supplemented
with 15% FBS, 2 mM PSG (Thermo Fisher), 50 ng/mL of
ascorbic acid 2-phosphatase (Sigma–Aldrich Corp.), and5 ng/mL of rhIL-7 (Peprotech). When expanding T cells,
5 ng/mL of rhIL-15 (Peprotech) was added to α-MEM.
All cell lines were cultured at 37 C with 5% CO2.

555

4.3 | Construction of retroviral vector
NZ-1 and NZ-27 scFv antibodies; Myc-Tag-inserted NZ-1
and NZ-27 scFv antibodies; CD8 transmembrane domain;and intracellular domain were amplified using polymerase chain reaction (PCR). KOD One PCR master mixblue (TOYOBO, Osaka, Japan) was used. PCR conditions
were as follows: denaturation for 2 min at 94 C, followed
by 35 cycles of denaturation at 98 C for 10 s and extension at 68 C for 15 s. Amplified genes were inserted into
the pMY retroviral vector (Cell BioLabs, San Diego, CA)
using NEBuilder HiFi Assembly (New England BioLabs,
Ipswich, MA). The primers are listed in Table S1.

4.4 | Generation of NZ-1 and NZ-27
CAR-T cells
GP293 cells were harvested on a 10-cm dish 2 days before
transfection. We transfected the CAR gene-encoded retrovirus vector and VSVG gene-encoded pMD2.G vector(Addgene) 2 days later using DNA transfection reagents.
For recombinant retroviral production, we generated
HT1080-based packaging cell lines reported previously(Iriguchi et al., 2021). For gene transduction, PBMCs
were stimulated with Dynabeads Human T-Activator
CD3/CD28 for T-cell expansion and activation (Thermo
Fisher). On days 2 and 3, activated T cells were loaded
onto 50 μg/mL of retronectin (Takara-bio, Kusatsu,
Japan)-coated 96-well plates that were preloaded withretrovirus supernatant. Retronectin-coated 96-well plates
were incubated at 4 C overnight. Retrovirus-preloaded
retronectin-coated 96-well plates were prepared bycentrifuging them for 2 h at 2000 g and 32 C.

4.5 | Flow cytometry analysis
We used an LSR or FACS AriaII flow cytometer(BD Biosciences, Franklin Lakes, NJ). These data wereanalyzed using the FlowJo software (Tree Star, Ashland,
OR). The antibodies used for staining are listed inTable S2. NZ-1 and NZ-27 scFvFc antibodies were developed as described previously (Chandramohan et al.,2013; Kaneko et al., 2020).

4.6 | Cytokine production
The BD Cytometric Bead Array Flex Set System(BD Biosciences) was used to measure human interleukin
2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor(TNF) levels. Cell culture supernatants were collected

556

after 24 h and centrifuged and frozen until use. Approximately 1.0  106 resting CAR-T cells were maintained in
α-MEM supplemented with 15% FBS, 2 mM PSG, 50 ng/
mL ascorbic acid 2-phosphatase, and 5 ng/mL rhIL-7,
and were cultured in the presence of 1.0  106 LN319,
PDPN-KO LN319, CHO/hPDPN, or CHO-K1 cells.

4.7 | Cytotoxicity assay
We used a nonradioactive cellular cytotoxicity assay kit(Techno-Suzuta, Nagasaki, Japan) according to the manufacturer's instructions. Effector cells were pre-enrichedwith CD8+ and TagBFP2+ T cells using the FACS AriaII
flow cytometer (BD Biosciences). Target cells (LN319,
PDPN-KO LN319, CHO-K1, CHO/hPDPN, and MG-63
cells) were pulsed with BM-HT reagent at 37 C for 15 min,washed three times, and seeded into 96-well plates at adensity of 5  103 cells/well. Effector cells were loaded
into the wells at effector-to-target (E:T) ratios ranging from
20:1 to 2.5:1 and were co-cultured for 3 h. Then, 40 μl of
the co-culture supernatant was mixed with 160 μl of Eu
solution, and time-resolved fluorescence was measured
using a NIVO plate reader (Perkin Elmer, Waltham, MA).
These cells were cultured in α-MEM supplemented with
15% FBS and 2 mM PSG at 37 C with 5% CO2.

ISHIKAWA ET AL.

expressing NZ-1 and NZ-27 CAR, we used Mem-PER™
Plus Membrane Protein Extraction Kit (Thermo Fisher)according to the manufacturer's protocol. In RIPA buffer
and protein extraction buffer included in Mem-PER™
Plus Membrane Protein Extraction Kit, Halt™ Phosphatase Inhibitor Cocktail (Thermo Fisher) was added, and5.0  106 Jurkat NFAT-luc cells were used on each assay.
Wes (protein simple, San Jose, CA) was used for observing
protein expression levels. On each assay, EZ Standard
Pack 1 (protein simple), Anti-Rabbit Detection Module forJess/Wes (protein simple), and Anti-Mouse Detection
Module for Jess/Wes (protein simple) were used according
to the manufacturer's protocol. Used antibodies were listed
in Table S2. Compass software for Simple Western (protein
simple) was used to evaluate each expression level.

4.10 | Luciferase assay
To obtain CAR-expressing Jurkat NFAT-luc cells, Jurkat
NFAT-luc cells were infected with retrovirus as describedabove. In total, 1.0  105 Jurkat NFAT-luc cells expressing
CAR and approximately 1.0  105 CHO/hPDPN and
CHO-K1 cells were co-cultured at 37 C for 3 h. Afterco-culture, luciferin (Promega) was added to evaluate
luciferase luminescence, measured using a NIVO plate
reader.

4.8 | Quantitative real-time PCR analysis
4.11 | In vivo animal models
The T-cell activation bioassay (NFAT, Promega, Madison,
WI) was used to evaluate CAR activation. Jurkat
NFAT-luc cells expressing CAR total RNA and complementary DNA were obtained using Sepasol-RNAISuper G(Nacalai Tesque, Kyoto, Japan) and the PrimeScript II 1st
strand cDNA synthesis kit (Takara-bio) according to manufacturers' instructions. Real-time quantitative PCR was
performed using StepOnePlus (Applied Biosystems, Waltham, MA). Designed primers and PowerUp SYBR green
master mix (Thermo Fisher) were used for PCR amplification. We used the standard cycling mode according to
the manufacturer's protocol. Expression levels of CAR
were normalized to those of glyceraldehyde 3-phosphate
dehydrogenase in each sample using the ΔΔCT method.

Six-week-old female NOD.Cg-PrkdcscidIL-2rgtm1Sug/ShiJic(NOG)_MHC-dKO mice were purchased from CIEA.
Approximately 5.0  106 LN319 cells mixed with BD
Matrigel matrix (growth factor reduced) (BD Biosciences) ina 3:5 ratio (v/v) were injected subcutaneously on day 0. In
total, 1.0  106 NZ-1 and NZ-27 CAR-positive T cells or
1.0  106 CAR-negative T cells were injected intravenously
on day 7. These CAR-T cells were infected with the retrovirus as described above. For T-cell expansion and activation,
they were injected into mice 7 days after stimulation with
Dynabeads human T-activator CD3/CD28. Tumor dimensions were measured with calipers, and tumor volumes were
calculated using the formula V = LW2/2, where L is thelength (longest dimension), and W is the width (shortest
dimension).

4.9 | Western blotting assay
To obtain the total protein of Jurkat NFAT-luc cells
expressing NZ-1 and NZ-27 CAR, we used RIPA Buffer(FUJIFILM Wako Pure Chemical Corporation) according
to the manufacturer's protocol. To obtain cytosol andplasma membrane protein of Jurkat NFAT-luc cells

4.12 | Analysis of tumor-infiltrating
iPS-CAR-T cells
NOG_MHC-dKO mice were sacrificed 14 days after T-cell
injection. Immunohistochemical staining was performed

ISHIKAWA ET AL.

at the Center for Anatomical, Pathological, and Forensic
Medical Research at Kyoto University. The anti-humanCD3 antibody (clone NCL-CD3-PS1; Leica Biosystems)
and Mouse to Mouse HRP staining System (ScyTek Lab.,UT) were used to stain the samples. Staining images were
captured using a fluorescence microscope (BZ-X800;
Keyence, Osaka, Japan).

4.13 | Statistical analyses
Data are represented as mean ± standard deviation ormean ± standard error of the mean. All statistical analyses were performed using GraphPad Prism 8 (GraphPad,
San Diego, CA) software. p-values were calculated using
one-way or two-way analysis of variance (ANOVA) with
Tukey's multiple comparison test and Student's t-test.
*p < .05, **p < .01, ***p < .005, and ****p < .001 wereconsidered statistically significant.
A C K N O WL E D G M E N T S
We thank the Center for Anatomical, Pathological, andForensic Medical Researches, Kyoto University, for their
help with immunohistochemical staining. We also thankMr Akito Tanaka, Ms Katsura Noda, and Ms Kanae
Mitsunaga (Kyoto University) for their technical andadministrative assistance. This research was supported by
the iPS Cell Research Fund.
FUNDING INFORMATIONThis work was supported by AMED (grant numbers
JP21bm1004001, JP21am0101078, and JP21am0401013).
CONFLICT OF INTEREST
Shin Kaneko is a founder, shareholder, and chief scientific officer at Thyas Co., Ltd. and received research funding from Takeda Pharmaceutical Co. Ltd., Kirin Co., Ltd.,
Astellas Co., Ltd., Tosoh Co. Ltd., and Thyas Co., Ltd.
ORCID
Akihiro Ishikawa https://orcid.org/0000-0002-76923851
Masazumi Waseda https://orcid.org/0000-0001-71795945
Mika K. Kaneko https://orcid.org/0000-0002-4158-9208
Yukinari Kato https://orcid.org/0000-0001-5385-8201
Shin Kaneko https://orcid.org/0000-0003-2291-4586
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SU PP O R TI N G I N F O RMA TI O N
Additional supporting information can be found online
in the Supporting Information section at the end of thisarticle.
How to cite this article: Ishikawa, A., Waseda,
M., Ishii, T., Kaneko, M. K., Kato, Y., & Kaneko, S.
(2022). Improved anti-solid tumor response byhumanized anti-podoplanin chimeric antigen
receptor transduced human cytotoxic T cells in an
animal model. Genes to Cells, 27(9), 549–558.
https://doi.org/10.1111/gtc.12972