MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 6, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0049
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A Defucosylated Mouse Anti-CD10 Monoclonal
Antibody (31-mG2a-f) Exerts Antitumor Activity
in a Mouse Xenograft Model of Renal Cell Cancers

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Hiroki Kawabata,1,* Tomokazu Ohishi,2,* Hiroyuki Suzuki,3 Teizo Asano,4 Manabu Kawada,2Hiroyoshi Suzuki,5 Mika K. Kaneko,4 and Yukinari Kato3,4

CD10 is a cell surface metalloendopeptidase that cleaves and degrades many secreted physiologically active
peptides by its enzymatic activity. Although CD10 expression has been found in various types of cells, its
expression is increased in several cancers, including renal cancer. In this study, the antitumor activity of a novelanti-human CD10 monoclonal antibody (mAb) was investigated. A defucosylated mouse IgG2a version of
C10Mab-31 (31-mG2a-f) was created from an anti-CD10 mAb, C10Mab-31 (IgG1, kappa). Both C10Mab-31 and
31-mG2a-f specifically reacted with endogenous CD10 in renal cancer cells, VMRC-RCW, with the dissociation
constant (KD) values of 6.3 · 10-9 M and 1.1 · 10-9 M, respectively, indicating high binding affinity. To further
examine the anti-CD10 mAb-mediated effector functions, the antibody-dependent cellular cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC) were examined. The 31-mG2a-f significantly exhibited ADCC
and CDC against VMRC-RCW cells in vitro. Furthermore, 31-mG2a-f exhibited antitumor activities in mouse
xenografts of VMRC-RCW cells. These results suggest that 31-mG2a-f exerts antitumor activities against
CD10-expressing renal cancers and could be a valuable therapeutic candidate for treating them.
Keywords: CD10, monoclonal antibody, ADCC, CDC, antitumor activity

Introduction

Renal cell carcinomas (RCCs), which originate fromthe renal epithelium of the renal tubules, account for
more than 90% of cancers in the kidney.(1) RCCs are the 6th
most common cancer among men and the 9th most common cancer among women in the United States,(2) and the 14th
most common cancer worldwide.(3) Treatment strategies forpatients with RCCs have been expanding recently with theadvent of immune checkpoint blockade by programmed cell
death-1 (PD-1)/PD-1 ligand 1 (PD-L1) inhibitors or cytotoxic
T lymphocyte-associated antigen 4 (CTLA-4) inhibitors.(4,5)
Although cytotoxic T cells play a vital role in antitumor
immunity, PD-1 negatively regulates the T cell function.

Its ligand PD-L1 is highly expressed by cancer cells; theinhibition of the PD-1–PD-L1 axis causes cytotoxic T cell
activation, which leads to tumor cell mortality. Similarly,
CTLA-4 also degrades cytotoxic T cell function; the inhibition of its function causes the T cell activation.(1) However,their therapeutic efficacy is limited, and more effective treatment options are required.
CD10 is a member of cell surface glycoproteins known asmembrane metalloendopeptidase, neprilysin, neutral endopeptidase, enkephalinase, EC3.4.24.11, and first identified
as a tumor-specific antigen (common acute lymphoblastic
leukemia antigen) in leukemia.(6–8) CD10 comprises thethree following domains: a short cytoplasmic N-terminal
domain, a transmembrane hydrophobic domain, and a large

1

Research and Development Center, Fuso Pharmaceutical Industries, Ltd., Osaka, Japan.
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Japan.
Departments of 3Molecular Pharmacology and 4Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai,
Japan.
5
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
*These two authors contributed equally to this work.
2320

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ANTITUMOR ACTIVITY OF ANTI-CD10 MAB

extracellular domain with catalytic activity. It is a membranebound zinc-dependent metalloprotease that degrades many
secreted physiologically active peptides, leading to the inactivation of several signaling pathways.(9) Altered expressionof CD10 has been seen in some hematological malignancies,
such as Burkitt lymphomas, acute lymphoblastic leukemia,
T and B cell lymphomas, and solid tumors.(8,10–12) Therefore,
CD10 has been used as useful diagnostic markers for leukemia, lymphoma, and various solid tumors.(8)Accumulating evidence suggests a role for CD10 in thedevelopment and prognosis of various tumors. Elevatedexpression of CD10 has been reported in multiple tumors,
including renal cancer,(13) lung cancer,(14) prostate cancer,(15,16)
papillary thyroid cancer,(17) head and neck cancer,(18) colorectal cancer,(19) esophageal cancer,(20) ovarian cancer,(21)and melanoma.(22) In addition, high expression of CD10 was
correlated with nodal metastasis, Gleason grade, and poor
prognosis of prostate cancer patients in the comparative
study.(15) Since CD10 is a cell surface metalloproteinase,increased expression of CD10 might facilitate the accumulation of inactivated peptides that are cleaved by CD10,
modulating the proliferation of cancer cells by influencing
the intracellular signal pathways in the tumor microenvironment.(23) In addition, CD10 also serves as a regulator in
the immune system.(7) These results suggest that CD10 canbe an attractive molecular target for cancer therapy.
In this study, a novel anti-human CD10 monoclonal antibody (mAb), C10Mab-31 (IgG1, kappa), was developed byimmunizing one mouse with the CD10-overexpressed humanrenal cancer OUR-10 cells. From the C10Mab-31, a defucosylated mouse IgG2a version of C10Mab-31 (31-mG2a-f) was
created. In this study, we evaluated whether 31-mG2a-f
induced antibody-dependent cellular cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC), or antitumor
activity against RCC cells in a mouse xenograft model.

321

100 units/mL of penicillin, 100 lg/mL of streptomycin, and0.25 lg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37C
in a humidified atmosphere containing 5% CO2 and 95% air.
Hybridoma production

A female 4-week-old BALB/c mouse was purchased from
CLEA Japan (Tokyo, Japan). Animals were housed under
specific pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved all animal experiments described in this study. A BALB/c mouse was
immunized using intraperitoneal injections of OUR-10/
CD10 (1 · 108 cells) together with Imject Alum (Thermo
Fisher Scientific, Inc.). After several additional immunizations, a booster injection was intraperitoneally administered
2 days before harvesting spleen cells. Spleen cells were thenfused with P3U1 cells using PEG1500 (Roche Diagnostics,
Indianapolis, IN). The resulting hybridomas were grown in
RPMI 1640 medium supplemented with hypoxanthine, aminopterin, and thymidine selection medium supplement(Thermo Fisher Scientific, Inc.). Culture supernatants werescreened using flow cytometry (CHO-K1 vs. CHO/CD10).
MAbs were purified from the supernatants of hybridomas,
cultured in Hybridoma-SFM medium (Thermo Fisher Scientific, Inc.) using Protein G-Sepharose (GE Healthcare
Biosciences, Pittsburgh, PA).
Production of the recombinant antibody

To generate 31-mG2a, we subcloned VH cDNA of C10Mab31 and CH of mouse IgG2a into the pCAG-Neo vector, alongwith cDNA of C10Mab-31 light chain into the pCAG-Zeo
vector, respectively. The vector of 31-mG2a was transfected into BINDS-09 cells (FUT8-KO ExpiCHO-S cells) using
the ExpiCHO Expression System (Thermo Fisher Scientific,Inc.). The resulting mAb, 31-mG2a-f, was purified using
Protein G-Sepharose.

Materials and Methods
Cell lines

P3X63Ag8U.1 (P3U1) and CHO-K1 cells were obtained
from the American Type Culture Collection (ATCC, Manassas, VA). OUR-10 and VMRC-RCW (renal cancer celllines) were provided from Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer,
Tohoku University (Miyagi, Japan). The pCAG-Ble vector
and the pCAG-Neo vector were purchased from FUJIFILM
Wako Pure Chemical Corporation (Osaka, Japan). OUR-10/
CD10 and CHO/CD10 cells were established by transfecting
pCAG-Ble/CD10-PAtag and pCAG-Neo/CD10-PAtag plasmids (Accession No. NM_000902.3) into OUR-10 cells and
CHO-K1 cells, respectively.
BINDS-27 (VMRC-RCW/CD10-knockout [KO] cells)
was established by transfecting CRISPR/Cas9 plasmids targeting human CD10 into VMRC-RCW cells (www.
med-tohoku-antibody.com/topics/001_paper_cell.htm). Using
TrueGuide gRNA tool, gRNA of CD10 was selected from
GeneArt predesigned gRNA database (Thermo Fisher Scientific, Inc., Waltham, MA). P3U1, VMRC-RCW, BINDS27, OUR-10, OUR-10/CD10, CHO-K1, and CHO/CD10
were cultured in RPMI 1640 medium (Nacalai Tesque, Inc.,
Kyoto, Japan), supplemented with 10% heat-inactivatedfetal bovine serum (FBS; Thermo Fisher Scientific, Inc.),

Flow cytometry

Cells were harvested by brief exposure to 0.25% trypsin/
1-mM ethylenediaminetetraacetic acid (EDTA; Nacalai
Tesque, Inc.). After washing with 0.1% bovine serum
albumin/phosphate-buffered saline (PBS), the cells weretreated with 1 lg/mL of anti-CD10 (C10Mab-31 and 31mG2a-f) for 30 minutes at 4C and subsequently with Alexa
Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA). Fluorescence data werecollected using EC800 Cell Analyzers (Sony Corp., Tokyo,
Japan).
Animals for ADCC and antitumor activities

All animal experiments were performed following relevant
guidelines and regulations to minimize animal suffering anddistress in the laboratory. Animal experiments for antitumor
activity were approved by the Institutional Committee forExperiments of the Institute of Microbial Chemistry (Permit
No. 2021-045 for ADCC assay and 2021-030 for antitumor
experiments). Mice were maintained in a specific pathogenfree environment (23C – 2C, 55% – 5% humidity) on an
11-hour light/13-hour dark cycle with food and water supplied ad libitum across the experimental period. Mice weremonitored for health and weight every 2–5 days during the322

3-week period of each experiment. We determined the lossof original body weight to a point >25% and/or a maximum
tumor size >3000 mm3 as humane endpoints for euthanasia.
Mice were euthanized by cervical dislocation; death was
verified by respiratory and cardiac arrest.
ADCC

suspended in DMEM with 10% FBS as effector cells. VMRCRCW or BINDS-27 cells were labeled with 10 lg/mL Calcein
AM (Thermo Fisher Scientific, Inc.) and resuspended in themedium. VMRC-RCW or BINDS-27 cells (2 · 104 cells/well) were placed in 96-well plates and mixed with effector
cells, 31-mG2a-f or control IgG (mouse IgG2a; Sigma-AldrichCorp., St. Louis, MO).
After a 4-hour incubation period, the Calcein release of
supernatant from each well was measured. The fluorescenceintensity was determined at an excitation wavelength of
485 nm and an emission wavelength of 538 nm using a
microplate reader (Power Scan HT; BioTek Instruments,
Winooski, VT). Cytolytic activity (as % of lysis) was
calculated using the following formula: % lysis = (E - S)/(M - S) · 100 (where E is the fluorescence released in the

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Six 6-week-old female BALB/c nude mice were purchasedfrom Charles River, and spleens were removed aseptically,and single-cell suspensions were obtained by dispersing the
spleens using a syringe and pressing through stainless steel
mesh. Erythrocytes were effectively lysed by 10-second
exposure to ice-cold distilled water. Splenocytes were washedwith Dulbecco’s modified Eagle’s medium (DMEM) and re-

KAWABATA ET AL.

FIG. 1. Production of 31-mG2a (mouse IgG2a) and 31-mG2a-f (defucosylated form) from an anti-CD10 mAb, C10Mab-31(mouse IgG1). (A) Schematic illustration of 31-mG2a and 31-mG2a-f. (B) Flow cytometry using C10Mab-31 and 31-mG2a-f.
VMRC-RCW and BINDS-27 cells were treated with C10Mab-31, 31-mG2a-f, or buffer control, followed by secondary
antibodies. (C) Determination of the binding affinity of C10Mab-31 and 31-mG2a-f for VMRC-RCW cells using flow
cytometry. VMRC-RCW cells were suspended in 100 mL serially diluted C10Mab-31 and 31-mG2a-f, followed by theaddition of Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were collected using the EC800 Cell Analyzer.
mAb, monoclonal antibody.

ANTITUMOR ACTIVITY OF ANTI-CD10 MAB

experimental cultures of target and effector cells, S is the
spontaneous fluorescence released in cultures containing
only target cells, and M is the maximum fluorescenceobtained by adding a lysis buffer containing 0.5% Triton
X-100, 10 mM Tris-HCl (pH 7.4), and 10 mM of EDTA tothe target cells to lyse all cells).
CDC

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VMRC-RCW or BINDS-27 cells were labeled with 10
lg/mL Calcein AM (Thermo Fisher Scientific, Inc.) andresuspended in the medium. VMRC-RCW or BINDS-27
cells (2 · 104 cells/well) were placed in 96-well plates and
10% of rabbit complement (Low-Tox-M Rabbit Complement;
Cedarlane Laboratories, Hornby, Ontario, Canada). After a
4-hour incubation period, the Calcein release of supernatantfrom each well was measured. Fluorescence intensity wascalculated as described in the above ADCC section.
Antitumor activity of 31-mG2a-f

323
Flow cytometry analysis against a renal cell cancer cellline, VMRC-RCW cells, using anti-CD10 mAbs

First, anti-CD10 mAbs were characterized using flow
cytometry. Both C10Mab-31 and 31-mG2a-f reacted with
VMRC-RCW cells, and not with BINDS-27 cells (VMRCRCW/CD10-KO cells), indicating that both C10Mab-31 and
31-mG2a-f are CD10-specific (Fig. 1B).
Kinetic analysis of the interactions of C10Mab-31 and 31mG2a-f with VMRC-RCW cells was conducted using flow
cytometry. As indicated in Figure 1C, the dissociation constant (KD) for the interaction of C10Mab-31 and 31-mG2a-f
with VMRC-RCW cells was 6.3 · 10-9 M and 1.1 · 10-9 M,
respectively. This suggests that both C10Mab-31 and 31mG2a-f exhibit high affinity for VMRC-RCW cells.
31-mG2a-f-mediated ADCC and CDC
in VMRC-RCW cells

Since the core-fucose-deficient anti-CD44 mAb (5-mG2a-f)
possesses ADCC and CDC activities in our previous study,(25)we next investigated whether 31-mG2a-f was capable ofFemale BALB/c nude mice (5-week old) were purchasedfrom Charles River (Kanagawa, Japan) and used in experiments when they were 7 weeks old. VMRC-RCW or BINDS27 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). A 100 lL suspension(containing 5 · 106 cells) was injected subcutaneously into
the left flanks of nude mice. After day 6, 100 lg of 31-mG2a-f
or control mouse IgG (Sigma-Aldrich Corp.) in 100 lL
PBS was injected into the peritoneal cavity of each mouse.
Additional antibodies were then injected on day 15. The
tumor diameter and volume were determined as previously
described.(24) The mice were euthanized 23 days after cell
implantation.
Statistical analyses

All data are expressed as mean – standard error of themean. Statistical analysis was conducted with Welch’s t-test
for ADCC, CDC, and tumor weight. ANOVA and Sidak’s
multiple comparisons tests were conducted for tumor volume
and mouse weight. All calculations were performed using
GraphPad Prism 8. A p-value of <0.05 was considered statistically significant.
Results
Development of anti-CD10 mAbs

In this study, one mouse was immunized with the OUR-10/
CD10 cells. Flow cytometry was conducted to check reactions with CHO-K1 and CD10-overexpressed CHO-K1(CHO/CD10) cells. A stronger reaction against CHO/CD10
was required compared with CHO-K1. One clone C10Mab-31of the IgG1 subclass was obtained. The subclass of C10Mab31 was converted into mouse IgG2a to add to the ADCC
and CDC activities. In addition, a defucosylated anti-CD10
mAb (31-mG2a-f) was produced using BINDS-09 cells(FUT8-KO ExpiCHO-S cells). This process is summarized
in Figure 1A.

FIG. 2. Evaluation of ADCC and CDC elicited by 31mG2a-f. (A) ADCC elicited by 31-mG2a-f or control mouse
IgG2a targeting VMRC-RCW or BINDS-27 cells. (B) CDC
elicited by 31-mG2a-f or control mouse IgG2a targeting
VMRC-RCW or BINDS-27 cells. Values are mean – SEM.
Asterisks indicate statistical significance (*p < 0.05; n.s., notsignificant; Welch’s t-test). ADCC, antibody-dependent
cellular cytotoxicity; CDC, complement-dependent cytotoxicity; SEM, standard error of the mean.

324

mediating ADCC against VMRC-RCW cells. As indicated in
Figure 2A, 31-mG2a-f showed ADCC (46% cytotoxicity)
against VMRC-RCW cells more effectively than it did against
control mouse IgG2a (36% cytotoxicity; p < 0.05). Contrastingly, 31-mG2a-f did not exert ADCC against BINDS-27 cells.
Whether 31-mG2a-f could mediate CDC against VMRCRCW cells was then investigated. As indicated in Figure 2B,
31-mG2a-f elicited a higher degree of CDC (52% cytotoxicity) in VMRC-RCW cells compared with that elicited by
the control mouse IgG2a (37% cytotoxicity; p < 0.05). Contrastingly, 31-mG2a-f did not exert CDC on BINDS-27 cells.
These results demonstrated that 31-mG2a-f promoted significantly higher levels of ADCC and CDC against VMRCRCW cells.

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Antitumor activities of 31-mG2a-f in the mouse
xenografts of VMRC-RCW cells

In the VMRC-RCW xenograft models, 31-mG2a-f (100 lg)
and control mouse IgG (100 lg) were injected intraperitoneally into mice on days 6 and 15 following the injection of

KAWABATA ET AL.

VMRC-RCW cells. The tumor volume was measured on days
6, 12, 15, 17, and 23 after the injection. The administrationof 31-mG2a-f resulted in a significant reduction in tumor
development on days 15 ( p < 0.05), 17 ( p < 0.01), and 23( p < 0.01) compared with that of the control mouse IgG(Fig. 3A). The administration of 31-mG2a-f resulted in 34%
reduction in tumor volume compared with that of the control mouse IgG on day 23.
In the BINDS-27 xenograft models, 31-mG2a-f (100 mg)
and control mouse IgG (100 mg) were also injected intraperitoneally into mice on days 6 and 15 following the injectionof BINDS-27 cells. The tumor volume of BINDS-27 cells
between the 31-mG2a-f-treated mice and control mouse IgGtreated mice showed no difference (Fig. 3B).
In the VMRC-RCW xenograft models, the tumor weightof the 31-mG2a-f-treated mice was significantly lower compared with the control mouse IgG-treated mice (17% reduction; p < 0.05, Fig. 3C). Contrastingly, the tumor weight
between the 31-mG2a-f-treated mice and control mouse IgGtreated mice indicated no difference in the BINDS-27 xenograft models (Fig. 3D).

FIG. 3. Evaluation of the antitumor activity of 31-mG2a-f in VMRC-RCW, or BINDS-27 xenografts. (A) VMRC-RCW
cells (5 · 106 cells) were injected subcutaneously into the left flank. After day 6, 100 mg of 31-mG2a-f or control mouse IgG
in 100 mL PBS was injected intraperitoneally into mice; additional antibodies were then injected on day 15. The tumor
volume was measured on days 6, 12, 15, 17, and 23 after the injection. Values are mean – SEM. Asterisks indicate statisticalsignificance (*p < 0.05, **p < 0.01, n.s., not significant; analysis of variance and Sidak’s multiple comparisons test).
(B) BINDS-27 cells (5 · 106 cells) were injected subcutaneously into the left flank. After day 6, 100 mg of 31-mG2a-f orcontrol mouse IgG in 100 mL PBS was injected intraperitoneally into mice; additional antibodies were then injected on day
15. The tumor volume was measured on days 6, 12, 15, 17, and 23 after the injection. Values are mean – SEM. n.s., notsignificant (analysis of variance and Sidak’s multiple comparisons test). (C) Tumors of VMRC-RCW xenografts were
resected from 31-mG2a-f and control mouse IgG groups. Tumor weight on day 23 was measured from excised xenografts.
Values are mean – SEM. Asterisk indicates statistical significance (*p < 0.05, Welch’s t-test). (D) Tumors of BINDS-27
xenografts were resected from 31-mG2a-f and control mouse IgG groups. Tumor weight on day 23 was measured fromexcised xenografts. Values are mean – SEM. n.s., not significant (Welch’s t-test). PBS, phosphate-buffered saline.

ANTITUMOR ACTIVITY OF ANTI-CD10 MAB

Mouse body weight was not significantly different among
the two groups in the VMRC-RCW or BINDS-27 xenograftmodels (Supplementary Fig. S1).
Altogether, these results indicate that the administrationof 31-mG2a-f effectively suppresses the tumor growth of
VMRC-RCW xenografts.

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Discussion

In immunotherapy, mAbs are recognized as ideal adjuvant therapeutic reagents for various human diseases.(26–28)
Because RCCs are highly vascular tumors, angiogenic factors, such as vascular endothelial growth factor-A (VEGF-A),
are overproduced in tumors.(29) Therefore, targeting the
VEGF signaling axis is an effective option for treating
RCCs.(30) The anti-VEGF-A mAb bevacizumab is usedwith interferon-a as a first-line option to treat patients with
RCCs.(31,32) In addition, several studies have investigated combinations of anti-VEGF therapy with other immune checkpoint blockade like PD-1/PD-L1, or CTLA-4 inhibitors.(1)CD10 is expressed in tumor cells and a wide variety ofnormal cells, including bone marrow stromal cells, granulocytes, a subset of B-cell progenitors, germinal center B cells,
and fibroblasts.(33) In a previous study, cancer-specific mAbs
(CasMabs) technology was established against cell surface
proteins, and the usefulness of CasMabs against tumors
was shown.(34,35) To prevent side effects of CD10-targeting
antibody therapy, the development of CasMabs is thought tohave a significant potential for an innovative therapeutic
approach. Since CasMabs could attack only cancer cells byrecognizing both target-derived peptide sequence and cancerspecific glycosylation, establishing CasMabs against humanCD10 would be an essential way to develop novel CD10targeting therapeutic antibodies without affecting normal
cells.
Recently, Mizutani et al. reported that a mouse anti-CD10
mAb ( JMAM-1) prolonged the survival in a tumor-bearing
mice model.(36) They showed that JMAM-1-treated malignant mesothelioma cell line MSTO-211H increased cell
cycle arrest, which is involved by cyclin-dependent kinase.
However, the precise molecular mechanisms about how
JMAM-1 affects antitumor activity remain unclear.
The main aim of this study was to investigate the antitumor activity of anti-CD10 mAb against RCC cells. The
antitumor effects of mAbs are mediated by several mechanisms of action, including ADCC and CDC.(25,37,38) Particularly, the ADCC activity plays a critical role of mAbs forcancer therapy, and natural killer cells are involved in the
immune system, exhibiting potent effector mechanisms
against cancer cells in humans.(39) It was demonstrated thata novel anti-CD10 mAb, C10Mab-31, and its defucosylatedmouse IgG2a version, 31-mG2a-f, specifically react with
RCC cells (Fig. 1). In addition, 31-mG2a-f significantly
exhibited ADCC and CDC in RCC cells in vitro (Fig. 2) andsuppressed tumor growth of the RCC cell xenograft modelin vivo (Fig. 3).
However, we could not determine whether 31-mG2a-f
affects the metalloprotease activity of CD10 and cross-reacts
with other species due to amino acid sequence homology. In
the future, we will examine the influence of our anti-CD10
mAbs on the CD10 enzymatic activity and determine thespecies cross-reactivity. Collectively, our findings in this325

study suggest that anti-CD10 mAbs could be a therapeuticcandidate, and the combined use with immune checkpoint
inhibitors might be a treatment option for patients with RCCs.
Acknowledgments

We would like to thank Ms. Akiko Harakawa [Instituteof Microbial Chemistry (BIKAKEN), Numazu, Microbial
Chemistry Research Foundation] for technical assistance inthe animal experiments.
Author Disclosure Statement

Y.K. received research funding from Fuso Pharmaceutical Industries, Ltd. The other authors have no conflict ofinterests.
Funding Information

This research was supported, in part, by Japan Agency
for Medical Research and Development (AMED) under
grant numbers JP22ama121008 (to Yukinari Kato),
JP21am0401013 (to Yukinari Kato), and JP21am0101078 (toYukinari Kato), and by the Japan Society for the Promotionof Science ( JSPS) Grants-in-Aid for Scientific Research(KAKENHI) grant numbers 21K07168 (to Mika K. Kaneko)
and 19K07705 (to Yukinari Kato).
Supplementary Material

Supplementary Figure S1
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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: October 7, 2021
Accepted: March 2, 2022