International Journal of

Molecular Sciences
ArticleAnti-HER2 Cancer-Specific mAb, H2Mab-250-hG1, Possesses
Higher Complement-Dependent Cytotoxicity than Trastuzumab
Hiroyuki Suzuki 1 , Tomokazu Ohishi 2,3 , Tomohiro Tanaka 1 , Mika K. Kaneko 1
1

2

3

*

Citation: Suzuki, H.; Ohishi, T.;
Tanaka, T.; Kaneko, M.K.; Kato, Y.
Anti-HER2 Cancer-Specific mAb,
H2 Mab-250-hG1 , Possesses Higher
Complement-Dependent Cytotoxicity
than Trastuzumab. Int. J. Mol. Sci.
2024, 25, 8386. https://doi.org/
10.3390/ijms25158386
Academic Editors: Yong-Seok Heo,
Hisashi Harada, Stergios Boussios andMatin Sheriffand Yukinari Kato 1, *Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.);
tomohiro.tanaka.b5@tohoku.ac.jp (T.T.); mika.kaneko.d4@tohoku.ac.jp (M.K.K.)
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
18-24 Miyamoto, Numazu-shi, Shizuoka 410-0301, Japan; ohishit@bikaken.or.jp
Institute of Microbial Chemistry (BIKAKEN), Laboratory of Oncology, Microbial Chemistry Research
Foundation, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
Correspondence: yukinari.kato.e6@tohoku.ac.jp; Tel.: +81-22-717-8207

Abstract: Cancer-specific monoclonal antibodies (CasMabs) that recognize cancer-specific antigens
with in vivo antitumor efficacy are innovative therapeutic strategies for minimizing adverse effects.
We previously established a cancer-specific anti-human epidermal growth factor receptor 2 (HER2)
monoclonal antibody (mAb), H2 Mab-250/H2 CasMab-2. In flow cytometry and immunohistochemistry, H2 Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal
epithelial cells. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, strongly recognizes
both breast cancer and normal epithelial cells in flow cytometry. The human IgG1 version of H2 Mab250 (H2 Mab-250-hG1 ) possesses compatible in vivo antitumor effects against breast cancer xenografts
to trastuzumab despite the lower affinity and effector activation than trastuzumab in vitro. This
study compared the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent
cellular cytotoxicity (CDC) between H2 Mab-250-hG1 and trastuzumab. Both H2 Mab-250-hG1 andtrastuzumab showed ADCC activity against HER2-overexpressed Chinese hamster ovary -K1 andbreast cancer cell lines (BT-474 and SK-BR-3) in the presence of human natural killer cells. Sometendency was observed where trastuzumab showed a more significant ADCC effect compared to
H2 Mab-250-hG1 . Importantly, H2 Mab-250-hG1 exhibited superior CDC activity in these cells compared to trastuzumab. Similar results were obtained in the mouse IgG2a types of both H2 Mab-250
and trastuzumab. These results suggest the different contributions of ADCC and CDC activities tothe antitumor effects of H2 Mab-250-hG1 and trastuzumab, and indicate a future direction for theclinical development of H2 Mab-250-hG1 against HER2-positive tumors.
Keywords: HER2; cancer-specific monoclonal antibody; antitumor effect; complement-dependent
cellular cytotoxicity

Received: 22 April 2024
Revised: 27 July 2024
Accepted: 30 July 2024
Published: 1 August 2024

Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/

1. Introduction
Human epidermal growth factor receptor 2 (HER2) is a member of the receptor tyrosine
kinases. Heterodimerization of HER2 with other HER family members and the ligands
or ligand-independent HER2 homodimerization results in the autophosphorylation of
the cytoplasmic domain. This event initiates a variety of signaling, such as the RAS andPI3K pathways, leading to cancer cell proliferation, survival, and invasiveness [1]. The
overexpression of HER2 is observed in approximately 20% of breast cancers [2] and 20%
of gastric cancers [3], which are associated with higher rates of recurrence and shorter
overall survival.
Trastuzumab, an anti-HER2 monoclonal antibody (mAb), exhibited in vitro antiproliferative efficacy and a potent antitumor effect in vivo [4,5]. The combination of4.0/).

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chemotherapy with trastuzumab in HER2-positive breast cancer patients with metastasis improves the progression-free survival and overall survival [6]. Trastuzumab wasapproved by the U.S. Food and Drug Administration (FDA) for the treatment of HER2positive breast cancer [6] and has been the most effective therapy for it for more than
20 years [7]. Trastuzumab is administered in patients with HER2-overexpressed tumors,which are defined by solid and complete membranous staining of more than 10% of cells in
immunohistochemistry (IHC 3+) and/or in situ hybridization (ISH)-amplified [8]. Furthermore, trastuzumab–deruxtecan (T-DXd), a trastuzumab-based antibody–drug conjugate(ADC), has been developed and approved by the FDA [9]. T-DXd exhibited superior
efficacy in not only HER2-positive breast cancers [10,11] but also HER2-low (IHC 1+ or IHC
2+/ISH-non-amplified) advanced breast cancers [12] and HER2-mutant lung cancers [13].
Because half of all breast cancers are classifiable as HER2-low, a significant number ofpatients are estimated to receive the benefit from T-DXd therapy [14].
The immunologic engagement of trastuzumab mediates the clinical efficacy [4]. Antibodydependent cellular cytotoxicity (ADCC) is elicited by natural killer (NK) cells or macrophages
upon the binding of Fcγ receptors (FcγRs) to the Fc region of mAbs [4]. Trastuzumab is ahumanized IgG1 mAb that binds to FcγRs [15] and activates macrophages, neutrophils, anddendritic cells, which change the adaptive immunity by cytokine production, chemotaxis,
and antigen presentation [4]. Moreover, the FcγR binding results in the activation of NK
cells and macrophages, which can result in the target cell killing [4]. However, the ADCC
is impaired by the N-linked glycosylation in the Fc region [16]. In particular, a lack of corefucose on the Fc N-glycan enhances the Fc binding to the FcγRs on effector cells [17]. Therefore, a core fucose deficiency on the Fc N-glycan has been shown to enhance the binding
to FcγR on effector cells [17] and exert potent antitumor effects [18]. The defucosylated
recombinant mAbs can be produced using fucosyltransferase 8-knockout Chinese hamster
ovary (CHO) cells [19].
Complement-dependent cellular cytotoxicity (CDC) is also exerted by the Fc domain
of mAbs [20,21]. Although complements have been thought of as an adjunctive component of the antibody-mediated cytolytic effects, complement is currently considered anessential effector of the tumor cytotoxic responses of mAb-based immunotherapy [21].
Through the development of a chimeric anti-CD20 mAb, rituximab, for the treatment of
B cell lymphomas, the involvement with the cytolytic capacity of the complement was
revealed in the antitumor effect [22,23]. In not only anti-CD20 but also anti-CD38 andCD52 immunotherapies, the cytolytic capacity of the tumor by complements has beenshown [23–25]. Furthermore, a growing body of evidence suggests that complements
play crucial functions in not only tumor cytolysis but also several immunologic roles
in antitumor immunity [26,27]. The crosstalk of complement effectors and cellular signaling pathways influence the T and B cell responses, T helper/effector T cell survival,
differentiation, and B cell activation.
A common adverse effect of anti-HER2 mAbs and the ADCs is cardiotoxicity [28].
Routine cardiac monitoring is required for patients [29]. Moreover, the lack of cardiac
trabeculae is observed in ErbB2 (ortholog of HER2)-knockout mice [30], and the featuresof dilated cardiomyopathy are observed in ventricular-specific ErbB2-knockout mice [31].
These results indicate that HER2 is involved in normal heart development and homeostasis.
Therefore, more selective or specific anti-HER2 mAbs against tumors are required to reduce
heart failures.
We previously developed cancer-specific anti-HER2 mAbs, H2 Mab-214/H2 CasMab1 [32] and H2 Mab-250/H2 CasMab-2 [33], from 278 clones of anti-HER2 mAbs using glioblastoma LN229-expressed HER2 as an antigen. Notably, both H2 Mab-214 and H2 Mab-250
did not react with spontaneously immortalized normal epithelial cells (HaCaT and MCF
10A) [32,33]. Moreover, H2 Mab-250 did not react with immortalized normal epithelial
cells derived from the mammary gland, lung bronchus, gingiva, kidney proximal tubule,
thymus, corneal, and colon [33]. In contrast, most anti-HER2 mAbs, including trastuzumab,
reacted with both cancer and normal epithelial cells [34]. The epitope mapping revealed

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that the Trp614 in the HER2 extracellular domain (ECD) 4 mainly contributes to the recognition by H2 Mab-250 [33]. H2 Mab-214 also recognized a similar epitope of H2 Mab-250,
and the crystal structure suggests that H2 Mab-214 recognizes a structurally misfolded
region in the HER2-ECD4, which usually forms a β-sheet [32]. The result indicates that the
local misfolding in the Cys-rich-ECD4 governs the cancer-specificity of H2 Mab-214. Furthermore, we produced mouse IgG2a -type and human IgG1 -type mAbs from H2 Mab-214
and H2 Mab-250. We found that both H2 Mab-214 and H2 Mab-250 possess a compatible
in vivo antitumor effect against breast cancer xenografts with trastuzumab despite thelower affinity and effector activation than trastuzumab in vitro [32,34].
This study compared the ADCC and CDC between H2 Mab-250 and trastuzumab
against HER2-overexpressed CHO-K1 (CHO/HER2) and breast cancer cell lines.
2. Results
2.1. ADCC and CDC of H2 Mab-250 and Trastuzumab against Breast Cancers
H2 Mab-250 recognized HER2-positive breast cancers (BT-474 and SK-BR-3) but did
not recognize HER2 in normal epithelial cells. In contrast, trastuzumab recognized bothtypes of HER2 [34]. Because H2 Mab-250 and trastuzumab are mouse IgG1 and human
IgG1 , respectively, we produced the human IgG1 type of recombinant H2 Mab-250 (H2 Mab250-hG1 ) and trastuzumab and confirmed the reactivity against breast cancers and normal
epithelial cells (Supplementary Figure S1). Although H2 Mab-250 possesses ~10-fold lower
affinity than trastuzumab, H2 Mab-250-hG1 possesses compatible antitumor effects against
BT-474 and SK-BR-3 xenografts compared to trastuzumab [34]. To reveal the contributionof the ADCC and CDC activities to the antitumor effects, we performed an in vitro ADCC
and CDC assay. Both BT-474 and SK-BR-3 cells were labeled with Calcein-AM. Then, thecalcein release was measured due to the cytotoxicity of mAbs plus human NK cell (ADCC)
or mAbs plus complements (CDC). As shown in Figure 1A, both H2 Mab-250-hG1 andtrastuzumab induced ADCC against BT-474 cells (47% and 64% cytotoxicity, respectively)
more effectively than the control human IgG (20% cytotoxicity; p < 0.01). Furthermore,
trastuzumab showed a superior ADCC compared to H2 Mab-250-hG1 (p < 0.01). In contrast,
H2 Mab-250-hG1 showed a significant CDC (47% cytotoxicity) compared to the control
human IgG (20% cytotoxicity; p < 0.05). However, trastuzumab did not show a significant
CDC (Figure 1B). In SK-BR-3, both H2 Mab-250-hG1 and trastuzumab induced ADCC
(12% and 17% cytotoxicity, respectively) more effectively than the control human IgG (4%cytotoxicity; p < 0.05 and p < 0.01, respectively, Figure 1C). Moreover, both H2 Mab-250-hG1
and trastuzumab exhibited CDC (42% and 26% cytotoxicity, respectively) more effectively
than the control human IgG (6% cytotoxicity; p < 0.01, Figure 1D). Importantly, H2 Mab250-hG1 showed a superior CDC compared to trastuzumab (p < 0.01, Figure 1D). These
results suggest the different contributions of ADCC and CDC to the antitumor effects of
H2 Mab-250-hG1 and trastuzumab.
2.2. ADCC and CDC by H2 Mab-250 and Trastuzumab against CHO/HER2
To confirm the requirement of HER2 in the ADCC and CDC of H2 Mab-250 andtrastuzumab, we used CHO-K1 and CHO/HER2 and performed the ADCC and CDC assays. CHO/HER2 was also recognized by H2 Mab-250 and trastuzumab with low and highreactivity, respectively [34]. As shown in Figure 2A, both H2 Mab-250-hG1 and trastuzumab
induced ADCC against CHO/HER2 cells (70% and 77% cytotoxicity, respectively) more
effectively than the control human IgG (13% cytotoxicity; p < 0.01). In contrast, H2 Mab250-hG1 showed a significant CDC (63% cytotoxicity) compared to the control human IgG(10% cytotoxicity; p < 0.01, Figure 2B). However, trastuzumab did not show a significant
CDC (Figure 2B). In CHO-K1, we did not observe ADCC and CDC in the presence of
H2 Mab-250 and trastuzumab (Figure 2C,D). These results indicate that the recognition of
HER2 is essential to the exertion of ADCC and CDC by H2 Mab-250-hG1 and trastuzumab.
Furthermore, dose-dependent activation of CDC activity by H2 Mab-250 and trastuzumab
was observed against CHO/HER2, but not CHO-K1 (Figure 2E). H2 Mab-250-hG1 exhibited

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Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW

4 of 16a significant CDC compared to the control human IgG from 25 µg/mL and showed it
compared to trastuzumab at 100 µg/mL (Figure 2E).

Figure 1. The
The ADCC
ADCC andand CDC
CDC activities
activities were mediatedtrastuzumab in
Figuremediated by
by H
H22Mab-250-hG
Mab-250-hG11 and trastuzumabin BTBT-474
SK-BR-3
cells.
(A,C)The
TheADCC
ADCCinduced
induced byin inthethepresenceof HofMab-250474
andandSK-BR-3
cells.
(A,C)
byhumanhumanNK
NKcells
cells
presenceH2
Mab-2502hG
orcontrol
controlhuman
humanIgG
IgG(hIgG)(hIgG)
againstBT-474
SK-BR-3(C) cells.
(B,D)
hG11,, trastuzumab,
trastuzumab, or
againstBT-474(A)(A)
andandSK-BR-3(C) cells.
(B,D)
The The
CDC
inducedbycomplements
inthepresence
of
100
µg/mL
of
H
2
Mab-250-hG
1,
trastuzumab,
CDC induced by complements in the presence of 100 µg/mL of H2 Mab-250-hG1 , trastuzumab, or orcontrolhumanIgG
IgGagainst
againstBT-474
BT-474(B)(B)
and
SK-BR-3
cells.
Valuesshown
as meanthe mean± SEM.
control human
andSK-BR-3(D)(D)
cells.
Valuesare areshown
as the± SEM.
Asterisks
indicate
statisticalsignificance
(**
p
<
0.01
and
*
p
<
0.05;
one-way
ANOVA
andTukey’s
Asterisks indicate statistical significance (** p < 0.01 and * p < 0.05; one-way ANOVA and Tukey’s
multiplecomparisonstest).
test).
multiple comparisons2.3. ADCC
Antitumor
by2Mab-250
H2 Mab-250-hG
1 and Trastuzumab
2.2.
andActivities
CDC by H
and Trastuzumab
against CHO/HER2
Next,
we examined
the in vivo antitumor
H2 Mab-250-hG
trastuzumab
1 andTo
confirm
the requirementof HER2efficacy
in the of
ADCC
and CDCof H
2Mab-250 andinthe
CHO/HER2
xenograftmodel.
We
injectedH
Mab-250-hG,
trastuzumab,
and as1
trastuzumab, we used CHO-K1 and CHO/HER2 and2 performed the
ADCC and CDC
control human IgG intraperitoneally on days 7, 14, and 21 after inoculating CHO/HER2.
says. CHO/HER2 was also recognized by H2Mab-250 and trastuzumab with low and highFurthermore, human NK cells were injected around the tumors on the same days of thereactivity, respectively [34]. As shown in Figure 2A, both H2Mab-250-hG1 andAbs injection. Following the inoculation, we measured the tumor volume on days 7, 14,
trastuzumab induced ADCC against CHO/HER2 cells (70% and 77% cytotoxicity, respec21, and 28. The H2 Mab-250-hG1 and trastuzumab administration led to a significant andtively)eﬀectively
than the control
human
p < 0.01).
contrast,
similarmore
reductionin the CHO/HER2
xenograftonIgG
day(13%28 (pcytotoxicity;< 0.01) compared
withInthatofH
2Mab-250-hG1 showed a significant CDC (63% cytotoxicity) compared to the control huthe control (Figure 3A). Both H2 Mab-250-hG1 and trastuzumab administration resulted inman
IgG(10% cytotoxicity;p < 0.01,
Figure 2B).
However,
trastuzumab
did notshow aan 81%
reductionin the CHO/HER2
xenograftvolumecomparedwith the control
humansignificant
IgG on dayCDC
28. (Figure 2B). In CHO-K1, we did not observe ADCC and CDC in the pres-ence of H2Mab-250 and trastuzumab (Figure 2C,D). These results indicate that the recognition of HER2 is essential to the exertion of ADCC and CDC by H2Mab-250-hG1 andtrastuzumab. Furthermore, dose-dependent activation of CDC activity by H2Mab-250 andtrastuzumab was observed against CHO/HER2, but not CHO-K1 (Figure 2E). H2Mab-250hG1 exhibited a significant CDC compared to the control human IgG from 25 µg/mL andshowed it compared to trastuzumab at 100 µg/mL (Figure 2E).

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2024, 25,
25, x8386

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of 16
15

Figure 2. The
The ADCC
ADCC andand CDC activities are mediatedand trastuzumab
trastuzumab in
Figuremediated by
by H
H22Mab-250-hG
Mab-250-hG11 andCHO/HER2
CHO/HER2andandCHO-K1
CHO-K1cells.
cells.(A,C)(A,C)The
TheADCC
ADCCinduced
inducedby
byhumanhumanNK
NK cells
cells inin thethe presence
presence of
CHO/HER2 (A)H
H22Mab-250-hG
Mab-250-hG11, ,trastuzumab,
trastuzumab,oror control
control human
human IgG
IgG (hIgG)(hIgG) against
against CHO/HER2(A) andand CHO-K1
CHO-K1(C)
cells.
(B,D)
The
CDC
inducedbycomplements
inthepresence
of
100
µg/mL
of
H
2Mab-250-hG1,(C) cells. (B,D) The CDC induced by complements in the presence of 100 µg/mL of H2 Mab-250trastuzumab, or control hIgG against CHO/HER2 (B) and CHO-K1 (D) cells. Values are shown as
hG1 , trastuzumab, or control hIgG against CHO/HER2 (B) and CHO-K1 (D) cells. Values are
the mean ± SEM. Asterisks indicate statistical significance (** p < 0.01; one-way ANOVA and Tukey’s
shown as the mean ± SEM. Asterisks indicate statistical significance (** p < 0.01; one-way ANOVA
multiple comparisons test). (E) The CDC induced by complements in the presence of 6.25, 25, andandµg/mL
Tukey’sofmultiple
comparisonstest). (E) The CDC induced by complements in the presence
1, trastuzumab, or hIgG. Values are shown as the mean ± SEM. Aster100
H2Mab-250-hG
of
6.25,
25,
and
100
µg/mL
of
H
Mab-250-hG
Valuesare shownas the1 , trastuzumab,
isks indicate statistical significance2 (** p < 0.01; two-way
ANOVA orandhIgG.
Tukey’smultiplecomparisonsmean ± SEM. Asterisks indicate statistical significance (** p < 0.01; two-way ANOVA and Tukey’stest).
multiple comparisons test).

Int. J. Mol. Sci. 2024, 25, 8386

treated CHO/HER2 xenograft-bearing mice (Figure 3D), and there was no diﬀerence inthe body appearance in those mice (Figure 3E).
We also investigated the pharmacokinetics of H2Mab-250-hG1 and trastuzumab afteradministration in nude mice. As shown in Supplementary Figure S2, the half-lives of
6 of 15
H2Mab-250-hG1 and trastuzumab were determined as 128 and 133 h, respectively. These
results indicate that H2Mab-250-hG1 possesses a similar half-life compared to trastuzumab.

Figure 3. Antitumor activity of H2 Mab-250-hG1 and trastuzumab against CHO/HER2 xenografts.
(A) CHO/HER2 cells (5 × 106 cells) were injected subcutaneously into the left flank of BALB/cnude mice (day 0). On day 7, 100 µg of H2 Mab-250-hG1 (n = 8), trastuzumab (n = 8), or control
human IgG (hIgG) (n = 8) was injected into the mice. On days 14 and 21, additional antibodies
were injected. Human NK cells were injected around the tumors on the same days as the Ab
administration (arrows). The tumor volume was measured on days 7, 14, 21, and 28. Values arepresented as the mean ± SEM. ** p < 0.01 (two-way ANOVA and Tukey’s multiple comparisons test).
The tumor weight (B) and appearance (C) of excised CHO/HER2 xenografts on day 28. Values arepresented as the mean ± SEM. * p < 0.05 (two-way ANOVA and Tukey’s multiple comparisons test).
The body weight (D) and appearance (E) of the xenograft-bearing mice treated with trastuzumab,
H2 Mab-250-hG1 , or a control hIgG.

The CHO/HER2 xenografts from the H2 Mab-250-hG1 - and trastuzumab-treated miceweighed significantly less than those from the control human IgG-treated mice (93% and
94% reduction, respectively; p < 0.05, Figure 3B,C). There was no significant difference
between the H2 Mab-250-hG1 - and trastuzumab-treated xenografts.
A body weight loss was not observed in the H2 Mab-250-hG1 - and trastuzumab-treated
CHO/HER2 xenograft-bearing mice (Figure 3D), and there was no difference in the body
appearance in those mice (Figure 3E).
We also investigated the pharmacokinetics of H2 Mab-250-hG1 and trastuzumab afteradministration in nude mice. As shown in Supplementary Figure S2, the half-lives of
H2 Mab-250-hG1 and trastuzumab were determined as 128 and 133 h, respectively. These
results indicate that H2 Mab-250-hG1 possesses a similar half-life compared to trastuzumab.

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2.4. ADCC and CDC by Mouse IgG2a -Type H2 Mab-250 and Trastuzumab against CHO/HER2
We next investigated the ADCC and CDC against CHO-K1 and CHO/HER2 using
mouse IgG2a -type H2 Mab-250 (H2 Mab-250-mG2a ) and trastuzumab (tras-mG2a ) to assess
the influence of the antibody format. As shown in Figure 4A, tras-mG2a induced ADCC
against CHO/HER2 cells (43% cytotoxicity) more effectively than the control mouse IgG2a(10% cytotoxicity; p < 0.05). In contrast, H2 Mab-250-mG2a did not significantly induce
ADCC against CHO/HER2 cells (Figure 4A). Furthermore, both H2 Mab-250-mG2a andtras-mG2a induced CDC against CHO/HER2 cells (51% and 45% cytotoxicity, respectively)
more effectively than the control mouse IgG2a (Figure 4B, 27% cytotoxicity; p < 0.01
andInt. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
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p < 0.05 respectively). In CHO-K1, we did not observe ADCC and CDC in the presence of
H2 Mab-250-mG2a and tras-mG2a (Figure 4C,D).

Figureactivities areare mediatedmediated by
by HH2 Mab-250-mG
2Mab-250-mG2a2a and tras-mG
tras-mG2a2a in
Figure 4. The
The ADCC
ADCC and CDC activities
CHO/HER2
andCHO-K1
cells.
(A,C)
The
ADCC
induced
by
human
NK
cells
in
the
presenceof
CHO/HER2 and CHO-K1 cells. (A,C) The ADCC induced by human NK cells in the presence100
ofMab-250-mG
2a, tras-mG2a, or control mouse IgG2a (mIgG2a) against CHO/HER2 (A) andµg/mLof H2of100 µg/mL
H2 Mab-250-mG
2a , tras-mG2a, or control mouse IgG2a (mIgG2a ) against CHO/HER2
CHO-K1 (C) cells. (B,D) The CDC induced by complements in the presence of H2Mab-250-mG2a,(A) and CHO-K1 (C) cells. (B,D) The CDC induced by complements in the presence of H2 Mab-250tras-mG2a, or control mIgG2a against CHO/HER2 (B) and CHO-K1 (D) cells. Values are shown as themG2a , tras-mG2a, or control mIgG2a against CHO/HER2 (B) and CHO-K1 (D) cells. Values are shown
mean± SEM. Asterisks indicate statistical significance (** p < 0.01 and * p < 0.05; one-way ANOVA
as themean multiple± SEM. Asterisks
indicate
andTukey’scomparisonstest). statistical significance (** p < 0.01 and * p < 0.05; one-way
ANOVA and Tukey’s multiple comparisons test).

2.5. Antitumor Activities by Mouse IgG2a-Type H2Mab-250 and Trastuzumab
These results indicate that tras-mG2a showed a superior ADCC compared to H2 MabNext,
we examined the in vivo antitumor eﬃcacy of H2Mab-250-mG2a and tras-mG2a
250-mG
2a . In contrast, H2 Mab-250-mG2a exhibited a superior CDC to CHO/HER2 cominthe
CHO/HER2
pared to tras-mG2a.xenograft model. We injected H2Mab-250-mG2a, tras-mG2a, and a control mouse IgG2a intraperitoneally on days 9 and 16 after inoculating CHO/HER2. Following the inoculation, we measured the tumor volume on days 9, 16, and 21. The H2Mab250-mG2a and tras-mG2a administration led to a potent and similar reduction in the
CHO/HER2 xenograft on days 16 and 21 (p < 0.01) compared with that of the control
mouse IgG2a (Figure 5A). The H2Mab-250-mG2a and tras-mG2a administration resulted in a
77% and 74% reduction in the CHO/HER2 xenograft volume compared with the control
mouse IgG2a on day 21.

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2.5. Antitumor Activities by Mouse IgG2a -Type H2 Mab-250 and Trastuzumab
Next, we examined the in vivo antitumor efficacy of H2 Mab-250-mG2a and tras-mG2ain the CHO/HER2 xenograft model. We injected H2 Mab-250-mG2a , tras-mG2a , and acontrol mouse IgG2a intraperitoneally on days 9 and 16 after inoculating CHO/HER2.
Following the inoculation, we measured the tumor volume on days 9, 16, and 21. The
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
9 of 16 in
H2 Mab-250-mG2a and tras-mG2a administration led to a potent and similar reduction
the CHO/HER2 xenograft on days 16 and 21 (p < 0.01) compared with that of the control
mouse IgG2a (Figure 5A). The H2 Mab-250-mG2a and tras-mG2a administration resulted in
a 77%
and 74%
reductionin the CHO/HER2
xenograftvolumemice.
compared
withhadthe nocontrol
wasobserved
betweenthe control
and H2Mab-250-mG
2a-treatedThose micemouse IgGin2abodyon day21.
diﬀerence
appearance(Figure 5E).

Figureactivityof of
H2Mab-250-mG
2a and tras-mG
2a againstagainst
xenografts.
(A)
Figure5.5.Antitumor
Antitumor
activity
H2 Mab-250-mG
CHO/HER2
xenografts.
2a and tras-mG
2a CHO/HER2
6 cells) were
CHO/HER2
cells
(5
×
10
injected
subcutaneously
into
the
left
flank
of
BALB/cnude
mice
6(A) CHO/HER2 cells (5 × 10 cells) were injected subcutaneously into the left flank of BALB/c(day 0). On day 9, 100 µg of H2Mab-250-mG2a (n = 8), tras-mG2a (n = 8), or a control mouse IgG2anude mice (day 0). On day 9, 100 µg of H Mab-250-mG (n = 8), tras-mG2a (n = 8), or a control
antibodies were injected (arrows). The(mIgG2a) (n = 8) was injected into mice. On day2 16, additional 2amousevolume
IgG2a (mIgG
wasinjected
into21.
mice.
On aredaypresented
16, additional
were2a ) (n = 8)ontumor
was measured
days
9, 16, andValuesas theantibodies
mean ± SEM.
** pinjected<(arrows).
The
tumor
volumewasmeasured
on
days
9,
16,
and
21.
Valuesarepresented
0.01 (two-way ANOVA and Tukey’s multiple comparisons test). The tumor weight (B) and appear-as themean(C)± of
SEM.
** pCHO/HER2< 0.01 (two-way
ANOVA
and
multiplecomparisonsThe
ance
excisedxenografts
on day21.Tukey’s
Values arepresented
as the meantest).
± SEM.
** tumorp<weight
0.01 (two-way
ANOVA
and
Tukey’smultiplecomparisonstest).
The
body
weight(D)
andappear(B) and appearance (C) of excised CHO/HER2 xenografts on day 21. Values are presentedance(E)meanof xenograft-bearing
withANOVA
H2Mab-250-mG
2a and tras-mG2a, or a control mIgG2a.
as the± SEM. ** p < mice
0.01 treated(two-way
and Tukey’s
multiple comparisons test). The
body weight (D) and appearance (E) of xenograft-bearing mice treated with H2 Mab-250-mG2a and2.6. Comparison of Antitumor Activities by H2Mab-250-hG1 and Trastuzumab in the Absence oftras-mG2a , or a control mIgG2a .

Human NK Cells

Asin Figure
3, we injected
NK cells 2awith
2Mab-250-hG
1 andmice
Theshown
CHO/HER2
xenografts
from thehumanH2 Mab-250-mG- andHtras-mG
2a -treatedtrastuzumab
because highactivitywasSince H
2Mab-250-hG
possesses
weighed significantly
less ADCC
than thosefromtheexpected.
control mouse
IgG(94% and2a -treated 1mice
a94%
higher
CDC activity,
we nextp compared
the antitumor
eﬀectsofno
H2Mab-250-hG
1 andreduction,
respectively;< 0.01, Figure
5B,C). Therewassignificant difference
trastuzumab
the human
NK cells. We injected H2Mab-250-hG1 and trastuzumab
between the without
H2 Mab-250-mG
2a - and tras-mG2a -treated xenografts.
intraperitoneallyon days7, 14,
andweight
21 afterloss
inoculating
the inoc- 2a Figure 5D shows
that
body
was notCHO/HER2.
observed inFollowing
the H2 Mab-250-mG
ulation,wemeasuredthetumor
volume
on
days
7,
10,
14,
16,
21,
24,
and
29.
We
observed
and tras-mG2a -treated CHO/HER2 xenograft-bearing mice. However, a slight difference
the more potent antitumor eﬃcacy of H2Mab-250-hG1 in relation to both the tumor volume(p < 0.01, Figure 6A) and weight (p < 0.01, Figure 6B) compared with that of trastuzumab
at day 29 (Figure 6C).
Body weight loss was not observed in the H2Mab-250-hG1- and trastuzumab-treated
CHO/HER2 xenograft-bearing mice (Figure 6D), and there was no diﬀerence in body ap-

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was observed between the control and H2 Mab-250-mG2a -treated mice. Those mice had nodifference in body appearance (Figure 5E).
2.6. Comparison of Antitumor Activities by H2 Mab-250-hG1 and Trastuzumab in the Absence of
Human NK Cells
As shown in Figure 3, we injected human NK cells with H2 Mab-250-hG1 and trastuzumab
because high ADCC activity was expected. Since H2 Mab-250-hG1 possesses a higher CDC
activity, we next compared the antitumor effects of H2 Mab-250-hG1 and trastuzumab without the human NK cells. We injected H2 Mab-250-hG1 and trastuzumab intraperitoneally ondays 7, 14, and 21 after inoculating CHO/HER2. Following the inoculation, we measured
the tumor volume on days 7, 10, 14, 16, 21, 24, and 29. We observed the more potent antituInt. J. Mol. Sci. 2024, 25, x FOR PEER mor
REVIEWof 16 6A)
efficacy of H2 Mab-250-hG1 in relation to both the tumor volume (p < 0.01,10
Figureand weight (p < 0.01, Figure 6B) compared with that of trastuzumab at day 29 (Figure 6C).

Figure 6.
1 and trastuzumab against CHO/HER2 xenografts
Figure
6. Antitumor
Antitumor activity
activityofofH
H2Mab-250-hG
2 Mab-250-hG1 and trastuzumab against CHO/HER2 xenografts
without human NK cell injection. (A) CHO/HER2 cells (5 × 106 cells)
were injected subcutaneously
without human NK cell injection. (A) CHO/HER2 cells (5 × 106 cells) were injected subcutaneously
into the left flank of BALB/c nude mice (day 0). On day 7, 100 µg of H2Mab-250-hG1 (n = 8) or
into
the left flank
BALB/cnude(dayOn0).daysOn day7, 100µg of H2 Mab-250-hG= 8) or1 (n intrastuzumab(n = 8)ofwas
injected
into micethe mice.
14 and21, additional
antibodies weretrastuzumab
(n
=
8)
was
injected
into
the
mice.
On
days
14
and
21,
additional
antibodies
were
injected
jected (arrows). The tumor volume was measured on days 7, 10, 14, 16, 21, 24, and 29. Values are(arrows).
tumor
on days
7, 10,and
14, Tukey’s
16, 21, 24,
and 29.comparisons
Values are presented
presentedTheas themeanvolume± SEM. was** p <measured
0.01 (two-way
ANOVA
multipletest).
Thethetumor
weight(B) and(C) of ANOVA
the excised
CHO/HER2
xenografts
on day 29. Valuesasmean± SEM.
** p appearance< 0.01 (two-way
andTukey’s multiple
comparisonstest). The
are presented
themean± SEM. ** (C)p < 0.01(two-way
and Tukey’s
multiplecomparisonstumor
weightas(B)andappearanceof theexcised ANOVACHO/HER2
xenografts
on day29. Values aretest). The body weight (D) and appearance (E) of the xenograft-bearing mice treated withpresented as the mean ± SEM. ** p < 0.01 (two-way ANOVA and Tukey’s multiple comparisons test).
trastuzumab or H2Mab-250-hG1.
The body weight (D) and appearance (E) of the xenograft-bearing mice treated with trastuzumab orH
Mab-250-hG1 .
3.2Discussion

We have developed CasMabs against HER2 (H2Mab-250 [33,34]), podocalyxin(PcMab-6 [35]), and podoplanin (LpMab-2 [36] and LpMab-23 [37]) by evaluating the reactivity against cancer and normal cells in flow cytometry and immunohistochemistry.
We also showed the in vivo antitumor eﬀect of the recombinant mAbs (mouse IgG2a orhuman IgG1 types) derived from the abovementioned mAbs [32–34]. Especially, H2Mab-

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Body weight loss was not observed in the H2 Mab-250-hG1 - and trastuzumab-treated
CHO/HER2 xenograft-bearing mice (Figure 6D), and there was no difference in body
appearance in those mice (Figure 6E).
3. Discussion
We have developed CasMabs against HER2 (H2 Mab-250 [33,34]), podocalyxin (PcMab6 [35]), and podoplanin (LpMab-2 [36] and LpMab-23 [37]) by evaluating the reactivity
against cancer and normal cells in flow cytometry and immunohistochemistry. We also
showed the in vivo antitumor effect of the recombinant mAbs (mouse IgG2a or human
IgG1 types) derived from the abovementioned mAbs [32–34]. Especially, H2 Mab-250
showed a potent antitumor effect in vivo [34] despite the lower reactivity and affinity
than trastuzumab in vitro [33]. However, the reason for this has not been clarified. In
this study, we compared the ADCC and CDC activity of H2 Mab-250 and trastuzumab
and found that H2 Mab-250 exhibited a superior CDC activity against breast cancer and
HER2-overexpressed cells compared to trastuzumab (Figures 1, 2 and 4). Furthermore, both
H2 Mab-250-hG1 and H2 Mab-250-mG2a showed compatible antitumor effects comparedto the corresponding isotype of trastuzumab (Figures 3 and 5). These results suggest that
the CDC activity of H2 Mab-250 would compensate for the lower ADCC activity in the
antitumor efficacy.
Complement is an essential effector of tumor cytotoxic responses in mAb-based immunotherapy [21]. The engagement of the Fc domain of mAbs with complement C1q
triggers the assembly of the active C1 complex (C1q, C1r, and C1s), which initiates thecascade. The downstream activation of terminal complement components results in theassembly of the pore-forming membrane attack complex (MAC or C5b–C9) on the tumor
cell membrane, which promotes the terminal lytic pathway [21]. Complement activation
also leads to tumor cell opsonization by C3-derived opsonins (C3b, iC3b, and C3dg), whichbind to the CR3/CR4 complement receptors on phagocytes (neutrophils and macrophages)
and augment the FcγR-dependent phagocytic uptake of opsonized tumor cells. Furthermore, complement activation generates pro-inflammatory mediators (C3a and C5a). The
anaphylatoxin C5a upregulates the FcγRs on phagocytes and primes them for enhanced
phagocytosis and increasing the magnitude of the tumor cytolytic response [21].
Because H2 Mab-250 showed increased CDC activity only in the presence of complement (Figures 1, 2 and 4), the assembly of MAC is thought to be efficiently formed on
the cells. Furthermore, the predisposition to CDC of H2 Mab-250 is independent of theisotype or species of mAbs (Figures 2 and 4). Therefore, the complementarity-determining
region and epitope of H2 Mab-250 are thought to be necessary. In Figure 6, the antitumor
effects of H2 Mab-250-hG1 were higher than those of trastuzumab without human NK cells,
indicating that H2 Mab-250-hG1 exerts antitumor activities with much higher CDC than
trastuzumab in vivo. Several factors, including the antigen size and density, determine
the engagement of the classical complement pathway. In addition, a geometry of theantigen–mAb complex allows efficient C1q binding [38]. Furthermore, IgG antibodies canform ordered hexamers upon binding to their antigen on cell surfaces. These hexamers
efficiently bind the hexavalent complement component C1q, the first step in the classicalpathway of complement activation [39,40]. The structure of the H2 Mab-250-HER2 complex
may provide adequate access for complements to exert CDC. Further investigation andconfirmation are required to clarify the mechanisms of CDC in H2 Mab-250.
Trastuzumab exerts antitumor activity through multiple mechanisms of action but isincapable of eliciting CDC in HER2-positive cancers in the presence of human serum [41,42].
As shown in Figure 1B, trastuzumab did not elicit CDC against BT-474 cells. An anti-HER2
bispecific and biparatopic antibody, zanidatamab, elicited potent CDC against HER2-hightumor cells, including BT-474 cells. Zanidatamab possesses an anti-HER2-ECD4 singlechain variable fragment (scFv) linked to heavy chain 1 and an anti-HER2-ECD2 fragment
antigen-binding (Fab) domain on heavy chain 2. Zanidatamab binds adjacent HER2
molecules in trans and initiates distinct HER2 reorganization and large HER2 clusters,

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which are not observed with trastuzumab [43]. Optimal CDC activity requires hexamericclustering of mAb Fc domains in the mAb–antigen clusters [44]. We identified the epitopeof H2 Mab-250 as 613- IWKFP-617 in the HER2-ECD4. The epitope of trastuzumab is a broader
sequence (residues 579-625), which includes the H2 Mab-250 epitope [33]. It is worthwhile
to investigate the ability of H2 Mab-250 to form a cluster with HER2.
Chimeric antigen receptor (CAR)-T cell therapy is rapidly advancing as a cancer
treatment; however, designing an optimal CAR remains challenging. Due to the specificreactivity against cancer cells, H2 Mab-250 is clinically developed as CAR-T cell therapy,
which is evaluated in a phase I study for HER2-positive advanced solid tumors in the US(NCT06241456). We discussed the benefit of reducing CAR affinity to limit trogocytosis,
which is observed in the high affinity of CAR-T cells [34]. In the monotherapy of H2 Mab-250,
we have reported compatible antitumor effects against breast cancer xenograft compared to
trastuzumab [32,34] and showed the importance of CDC in this study. Extensive research
indicates that resistance to CDC is induced by the expression of complement regulators
in tumor cells during the escape from host immune responses. Notably, an upregulationof the regulators, including CD46, CD55, and CD59, has been shown to prevent CDC
through suppression of terminal complement activation and MAC assembly [45–47]. In
this regard, several strategies have been developed to overcome the resistance to CDC inmAb-based immunotherapy [48–51]. Therefore, dual targeting of HER2 by H2 Mab-250 andcomplement regulators should be investigated in future studies in in vitro models.
4. Materials and Methods
4.1. Cell Culture
Chinese hamster ovary (CHO)-K1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). CHO-K1 and HER2-overexpressed CHO-K1(CHO/HER2) [33] were cultured in the Roswell Park Memorial Institute (RPMI)-1640
medium (Nacalai Tesque, Inc., Kyoto, Japan). BT-474 and SK-BR-3 were also obtained
from the ATCC, and they were cultured in Dulbecco’s Modified Eagle Medium (DMEM)(Nacalai Tesque, Inc.). These media were supplemented with 10% fetal bovine serum (FBS;
Thermo Fisher Scientific Inc., Waltham, MA, USA), antibiotic–antimycotic mixed solution(Nacalai Tesque, Inc.). All the cell lines were cultured at 37 ◦ C in a humidified atmospherewith 5% CO2 and 95% air.
4.2. Production of Recombinant mAbs
To generate H2 Mab-250-hG1 , VH of H2 Mab-250 and CH of human IgG1 were cloned
into the pCAG-Ble vector. The VL of the H2 Mab-250 and CL of the human kappa lightchain were cloned into the pCAG-Neo vector.
To generate H2 Mab-250-mG2a , we cloned the VH cDNA of H2 Mab-250 and CH ofmouse IgG2a into the pCAG-Ble vector. To generate a mouse IgG2a type of trastuzumab(tras-mG2a ), the VH cDNA of trastuzumab and the CH cDNA of mouse IgG2a were cloned
into the pCAG-Neo vector, and the VL cDNA of trastuzumab and the CL cDNA of mouse
kappa light chain were cloned into the pCAG-Ble vector.
To generate the control mouse IgG2a (PMab-231), we cloned the heavy and light chainsof PMab-231 [52] into the pCAG-Neo and pCAG-Ble vectors, respectively.
The vectors were transfected into BINDS-09 (fucosyltransferase 8-knockout ExpiCHOS; http://www.med-tohoku-antibody.com/topics/001_paper_cell.htm, accessed on 20
April 2024) cells using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc.)
to produce the defucosylated mAbs. The H2 Mab-250-mG2a , tras-mG2a , H2 Mab-250-hG1 ,trastuzumab, and PMab-231 were purified using Ab-Capcher (Kagawa, Japan). Afterwashing with PBS, the bound antibodies were eluted with an IgG elution buffer (Thermo
Fisher Scientific Inc.) and immediately neutralized using 1 M Tris-HCl (pH 8.0). Finally,
the eluates were concentrated using Amicon Ultra (Merck KGaA) and replaced with PBS.
The purified mAbs were confirmed by SDS-PAGE in reduced and non-reduced conditions(Supplementary Figure S3).

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Normal human IgG was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA).
4.3. ADCC
The ADCC of H2 Mab-250-hG1 and trastuzumab was measured as follows. HumanNK cells were purchased from Takara Bio, Inc. (Shiga, Japan) and were used as effector
cells. The NK cells were used in the following experiment immediately after thawing.
We labeled the target cells (BT-474, SK-BR-3, CHO-K1, and CHO/HER2) using 10 µg/mL
Calcein AM (Thermo Fisher Scientific, Inc.). The target cells were plated in 96-well plates(1 × 104 cells/well) and mixed with the human NK cells (effector to target ratio, 50:1) and100 µg/mL of H2 Mab-250-hG1 , trastuzumab or control human IgG. The calcein release wasmeasured after a 4.5 h incubation, and the cytotoxicity (% lysis) was calculated as described
previously [34].
The ADCC of H2 Mab-250-mG2a and tras-mG2a was measured as follows. Effector cells
were obtained from the spleen of female BALB/c nude mice (Jackson Laboratory Japan,
Inc., Kanagawa, Japan). The Calcein AM-labeled target cells (CHO-K1 and CHO/HER2)
using 10 µg/mL Calcein AM were plated in 96-well plates (1 × 104 cells/well) and mixed
with the effector cells (effector to target ratio, 50:1) with 100 µg/mL of H2 Mab-250-mG2aand tras-mG2a or control mouse IgG2a . After a 4 h incubation at 37 ◦ C, the calcein release
into the medium was measured, and the cytotoxicity (% lysis) was calculated.
4.4. CDC
The calcein-labeled target cells (BT-474, SK-BR-3, CHO-K1, and CHO/HER2) wereplated and mixed with rabbit complement (final dilution 1:10, or 1:15 [Figure 2E], Low-ToxM Rabbit Complement; Cedarlane Laboratories, Hornby, ON, Canada) and the indicatedconcentration of H2 Mab-250-hG1 , trastuzumab or control human IgG. Following incubationfor 4.5 h at 37 ◦ C, the calcein released into the medium was measured, as described above.
In the case of H2 Mab-250-mG2a and tras-mG2a or control mouse IgG2a , we performed a 4 h
incubation at 37 ◦ C and the cytotoxicity (% lysis) was calculated.
4.5. Antitumor Activities of H2 Mab-250-hG1 , Trastuzumab, H2 Mab-250-mG2a , and Tras-mG2a , inTumor Xenograft Models
To examine the antitumor effect of H2 Mab-250-hG1 , trastuzumab, H2 Mab-250-mG2a ,and tras-mG2a , animal experiments were approved by the Institutional Committee forExperiments of the Institute of Microbial Chemistry (approval no. 2023-066 and 2023-074).
We determined the body weight loss exceeding 25% and maximum tumor size exceeding3000 mm3 as humane endpoints.
CHO/HER2 cells were suspended in BD Matrigel Matrix Growth Factor Reduced(BD Biosciences, San Jose, CA, USA). Then, BALB/c nude mice (Jackson Laboratory Japan,
Kanagawa, Japan) were injected subcutaneously in the left flank with 100 µL of the suspension (5 × 106 cells). On days 9 and 16 post-inoculation, 100 µg of H2 Mab-250-mG2a(n = 8), tras-mG2a (n = 8), or control mouse IgG2a (PMab-231; n = 8) in 100 µL PBS was
intraperitoneally injected.
For evaluation of H2 Mab-250-hG1 and trastuzumab, we injected the mice with 100 µgof H2 Mab-250-hG1 (n = 8), trastuzumab (n = 8), or control human IgG (n = 8) in 100 µL
of PBS through intraperitoneal injection on days 7, 14, and 21 post-inoculation. Furthermore, human NK cells (8.0 × 105 cells, Takara Bio, Inc.) were injected near the tumors
subcutaneously on same days as the mAbs injection.
The tumor volume was calculated using the following formula: volume = W2 × L/2,
where W is the short diameter and L is the long diameter. All the mice were euthanized bycervical dislocation.
Statistical analyses were performed using GraphPad PRISM 6 (GraphPad Software,Inc., La Jolla, CA, USA).

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4.6. Pharmacokinetics of H2 Mab-250-hG1 and Trastuzumab
HER2 ectodomain [34] was immobilized on Nunc Maxisorp 96-well immunoplates(Thermo Fisher Scientific Inc.) at a concentration of 1 µg/mL for 30 min at 37 ◦ C. Afterwashing with PBS containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.), the wells
were blocked with 1% (w/v) bovine serum albumin (BSA)-containing PBST for 30 min at
37 ◦ C. To make a standard curve, the serially diluted H2 Mab-250-hG1 and trastuzumab(0.00064–10 µg/mL) were added to each well, followed by peroxidase-conjugated antihuman Fc (1:3000 diluted; Sigma-Aldrich Corp.). The enzymatic reactions were conductedusing ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.), followed by the measurementof the optical density at 655 nm, using an iMark microplate reader (Bio−Rad Laboratories,
Inc., Berkeley, CA, USA). The standard curve was produced using GraphPad PRISM 6.
H2 Mab-250-hG1 and trastuzumab (100 µg/mouse, n = 3) were intraperitoneally injected
and the serums were collected from day 0 (4 h after injection) to 10. The concentration ofmAbs was determined as described above. The half-life of the mAbs was calculated as
described previously [53].
Supplementary Materials: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms25158386/s1.
Author Contributions: H.S., T.O. and T.T. performed the experiments. M.K.K. and Y.K. designed theexperiments. H.S., T.T. and Y.K. analyzed the data. H.S. and Y.K. wrote the manuscript. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was supported in part by the Japan Agency for Medical Research andDevelopment (AMED) under the following grant numbers: 24am0521010 (to Y.K.), JP23ama121008(to Y.K.), JP23am0401013 (to Y.K.), JP23bm1123027 (to Y.K.), JP23ck0106730 (to Y.K.), JP18am0301010(to Y.K.), and JP21am0101078 (to Y.K.).
Institutional Review Board Statement: The Institutional Committee for Experiments of the Instituteof Microbial Chemistry approved the animal experiment to examine the antitumor effect (approval
no. 2023-066 and 2023-074).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article.
Conflicts of Interest: The authors declare no conflicts of interest.

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