Article

Development of a Novel Anti-CD44 Variant 8 Monoclonal
Antibody C44Mab-94 against Gastric Carcinomas
Hiroyuki Suzuki 1,2,*,†, Nohara Goto 1,†, Tomohiro Tanaka 1, Tsunenori Ouchida 2, Mika K. Kaneko 1,2
and Yukinari Kato 1,2,*
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; s1930550@s.tsukuba.ac.jp (N.G.);
tomohiro.tanaka.b5@tohoku.ac.jp (T.T.); k.mika@med.tohoku.ac.jp (M.K.K.)
2 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; tsunenori.ouchida.d5@tohoku.ac.jp* Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-22-717-8207 (H.S. & Y.K.)† These authors contributed equally to this work.
1

Citation: Suzuki, H.; Goto, N.;
Tanaka, T.; Ouchida, T.; Kaneko, M.K.;

Abstract: Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC
with peritoneal metastasis exhibits a poor prognosis due to the lack of effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed intumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeted monoclonal antibodies (mAbs) is importantfor GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed
PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of
the clones (C44Mab-94; IgG1, kappa) recognized the variant-8-encoded region and peptide, indicating that C44Mab-94 is a specific mAb for CD44v8. Furthermore, C44Mab-94 could recognize
CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and
NUGC-4) in flow cytometric analyses. C44Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastriccancer cells. These results indicate that C44Mab-94 is useful for detecting CD44v8 in a variety ofexperimental methods and is expected to become usefully applied to GC diagnosis and therapy.

Kato, Y. Development of a Novel
Anti-CD44 Variant 8 Monoclonal
Antibody C44Mab-94 against Gastric

Keywords: CD44 variant 8; monoclonal antibody; gastric cancer; flow cytometry;immunohistochemistry

Carcinomas. Antibodies 2023, 12, 45.
https://doi.org/10.3390/
antib12030045
Academic Editor: Wei Li
Received: 9 May 2023
Revised: 28 June 2023
Accepted: 3 July 2023
Published: 4 July 2023

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

1. Introduction
Gastric cancer (GC) is the third leading cause of cancer-related deaths globally [1].
The GC incidence is higher in Eastern Asia than in Western countries [2]. The vast majority
of GC are adenocarcinomas, which can be divided into intestinal-type gastric cancer(IGC), diffuse-type gastric cancer (DGC), and mixed histology according to the Laurenclassification [3]. The World Health Organization classifies gastric adenocarcinomas into
papillary, tubular, mucinous, and poorly cohesive carcinomas [4]. Furthermore, next-generation sequencing defined four molecular subtypes, including Epstein–Barr virus-positive, microsatellite instability, genomically stable, and chromosomally unstable types[5,6]. The analysis also revealed the alterations in the GC genome and provided treatment
options with anti-human epidermal growth factor receptor 2 (HER2) therapy [7] or immune checkpoint inhibitor therapy [8]. However, the benefit of those therapies is limited
to a small subset of patients. In patients with advanced GC, especially those with DGC,
peritoneal metastasis and subsequent development of malignant ascites are the most

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Antibodies 2023, 12, 45

2 of 17frequent cause of death [9]. Tanaka et al., therefore, performed a comprehensive multiomic analysis of malignant ascitic samples and their corresponding tumor cell lines [10].
They identified the genomic alterations and significant amplification of known cancer
driver genes, such as KRAS, FGFR2, MET, ERBB2, EGFR, MYC, CCND1, and CD44 in GC
with peritoneal metastasis [10]. Among them, the cell surface antigens (FGFR2, MET,
HER2, EGFR, and CD44) are potentially treatable with monoclonal antibody (mAb) therapy [11]. In contrast to the first four antigens, neither therapy nor diagnosis against CD44
with mAb has yet been established.
CD44 plays important roles in tumor progression and has various isoforms [12],which are generated by the alternative splicing of CD44 mRNA [13]. The mRNA of theCD44 standard (CD44s) isoform is produced by constant region exons including the first
five (referred to as exons 1 to 5) and the last five (16 to 20) [14]. The mRNAs of CD44 variant(CD44v) isoform are produced by the assembling of variant exons (v1–v10) with the constantregion exons of CD44s [15]. CD44s and CD44v receive post-translational modifications, suchas N-glycosylation and O-glycosylation [16]. Therefore, the molecular weight of CD44s is
80~100 kDa, while CD44v has various molecular weights (100~250 kDa) due to a variety ofglycosylation [17]. Both CD44s and CD44v (pan-CD44) can attach to hyaluronic acid (HA),
which is important for cellular adhesion, homing, and motility [18].
CD44v is overexpressed in tumors [12] and promotes tumor malignant progression
through the binding to growth factors and the acquisition of invasiveness, stemness, anddrug resistance [19–21]. These were mediated by the unique functions of the variant’s
exon-encoded region. The v3-encoded region can recruit heparin-binding growth factors
to their receptor and promote the signal transduction [22,23]. The v6-encoded region
forms a ternary complex with hepatocyte growth factor and its receptor MET, which isessential for the activation [24,25]. Furthermore, the v8–10-encoded region binds to and stabilizes a cystine–glutamate transporter (xCT), which enhances cystine uptake and glutathione
synthesis [26]. The elevation of reduced glutathione (GSH) mediates the defense against reactive oxygen species (ROS) [26] and chemotherapeutic drugs [27]. The expression of CD44v8–
10 is associated with the function of xCT and intracellular redox status, which in turn is associated with poor prognosis [28]. Therefore, the establishment of CD44v-specific mAbs is essential for CD44-targeted tumor diagnosis and therapy. However, the roles played in tumor
development by the variant 8-encoded region have not been fully elucidated.
Our group previously established an anti-pan-CD44 mAb—C44Mab-5 (IgG1, kappa)[29]—using the Cell-Based Immunization and Screening (CBIS) method. Moreover, another anti-pan-CD44 mAb—C44Mab-46 [30]—was developed by immunizing mice with
CD44v3–10 ectodomain. C44Mab-5 and C44Mab-46 have epitopes within the standard exon
2 and 5-encoding regions, respectively [31,32]. We further showed that both C44Mab-5 andC44Mab-46 are applicable to flow cytometry and immunohistochemical analyses in oral
squamous cell carcinomas (OSCC) [29] and esophageal SCC [30]. Furthermore, we produced a class-switched and a defucosylated version of C44Mab-5 (5-mG2a-f) using fucosyltransferase 8-deficient ExpiCHO-S cells and evaluated the antitumor effects of 5-mG2a-f in
OSCC xenograft-bearing mice [33]. We have developed various anti-CD44v mAbs, including anti-CD44v4 (C44Mab-108) [34], anti-CD44v5 (C44Mab-3) [35], anti-CD44v6 (C44Mab-9)[36], anti-CD44v7/8 (C44Mab-34) [37], and anti-CD44v9 (C44Mab-1) [38].
In this study, we established a novel anti-CD44v8 mAb—C44Mab-94 (IgG1, kappa)—
using the CBIS method and evaluated its applications.
2. Materials and Methods
2.1. Cell Lines
The human OSCC cell line (HSC-3) and the human gastric cancer cell lines (MKN45
and NUGC-4) were obtained from the Japanese Collection of Research Bioresources(Osaka, Japan). The human pancreatic cancer cell line (PANC-1) was obtained from the
Cell Resource Center for Biomedical Research Institute of Development, Aging, andAntibodies 2023, 12, 45

3 of 17Cancer at Tohoku University (Sendai, Japan). Chinese hamster ovary (CHO)-K1 andP3X63Ag8U.1 (P3U1; a mouse multiple myeloma) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HSC-3 was cultured in DMEM
medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 100 μg/mL streptomycin, 100 U/mL penicillin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.), and 10% (v/v)
heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA).
The cell lines (MKN45, NUGC-4, PANC-1, CHO-K1, and P3U1) were cultured in RPMI-1640
medium (Nacalai Tesque, Inc.), supplemented as indicated above. All cells were cultured using a humidified incubator at 37 °C, in an atmosphere of 5% CO2 and 95% air.
2.2. Construction of Plasmid DNA and Establishment of Stable Transfectants
The cDNAs of CD44s and CD44v3–10 were obtained as described previously [29].
The cDNAs were cloned into pCAG-zeo-ssPA16 and pCAG-neo-ssPA16 vectors with asignal sequence and N-terminal PA16 tag (GLEGGVAMPGAEDDVV). The PA16 tag canbe detected by NZ-1 mAb, which was originally developed as an anti-human podoplanin(PDPN) mAb. N-terminal PA16-tagged CD44v3–10 deletion mutants (dN224, dN266,
dN304, dN343, dN386, dN430, dN464, dN494, and dN562) were produced using a HotStar
HiFidelity Polymerase Kit, and subcloned into the pCAG-neo-ssPA16 vector. The pCAGzeo-ssPA16-CD44s, pCAG-zeo-ssPA16-CD44v3–10, and pCAG-neo/PA16-CD44v3–10 deletion mutant vectors were transfected into CHO-K1 cells. The pCAG-neo/PA16-CD44v3–
10 vector was transfected into PANC-1 cells. The transfection was performed using a Neontransfection system (Thermo Fisher Scientific, Inc.). By the limiting dilution method, stabletransfectants PANC-1/CD44v3–10, CHO/CD44s, CHO/CD44v3–10, and several deletionmutants of CHO/CD44v3–10 (dN224, dN266, dN304, dN343, dN386, dN430, dN464,
dN494, and dN562) were finally established.
2.3. Production of Hybridomas
The 6-week-old female BALB/c mice were purchased from CLEA Japan (Tokyo, Japan). The mice were intraperitoneally immunized with PANC-1/CD44v3–10 (1 × 108 cells)
and Imject Alum (Thermo Fisher Scientific Inc.). Additional immunizations of PANC1/CD44v3–10 (1 × 108 cells, three times) and a booster injection of PANC-1/CD44v3–10 (1× 108 cells) was performed 2 days before the sacrifice. Hybridomas were produced as described previously [35]. The supernatants, which are positive for CHO/CD44v3–10 cells
and negative for CHO-K1 cells, were selected by flow cytometry, SA3800 Cell Analyzers(Sony Corp., Tokyo, Japan).
2.4. Enzyme-Linked Immunosorbent Assay (ELISA)
Four peptides, covering the v7, v8, and v9 regions of CD44v3–10, were obtained from
Sigma-Aldrich Corp. (St. Louis, MO, USA).
The peptide sequences were as follows.
CD44p421–440 (GHQAGRRMDMDSSHSTTLQP); v7/v8,
CD44p431–450 (DSSHSTTLQPTANPNTGLVE); v8,
CD44p441–460 (TANPNTGLVEDLDRTGPLSM); v8,
CD44p451–470 (DLDRTGPLSMTTQQSNSQSF); v8/v9.
The peptides (10 μg/mL) were immobilized on 96-well immunoplates (Nunc Maxisorp; Thermo Fisher Scientific Inc). The immunoplate washing was performed with PBS
containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.). The blocking was performed
with 1% (w/v) bovine serum albumin (BSA) in PBST. C44Mab-94 (10 μg/mL) or blocking
buffer was added to the peptide-coated wells. Next, the wells were further incubated with
anti-mouse immunoglobulins conjugated with peroxidase (1:2000 dilution; Agilent Technologies Inc., Santa Clara, CA, USA). The ELISA POD Substrate TMB Kit (Nacalai Tesque,Inc.) was used for the peroxidase reaction. Using an iMark microplate reader (Bio-Rad
Laboratories, Inc., Berkeley, CA, USA), the optical density (655 nm) was measured.

Antibodies 2023, 12, 45

4 of 172.5. Flow Cytometry
HSC-3, MKN45, NUGC-4, CHO-K1, CHO/CD44s, CHO/CD44v3–10, andCHO/CD44v3–10 deletion mutants were obtained using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). In the CBIS screening andepitope mapping, the hybridoma supernatants were treated with CHO-K1,
CHO/CD44v3–10, or CHO/CD44v3–10 deletion mutants. In the dose-dependent assay, thecells were incubated with C44Mab-94, C44Mab-46, or control blocking buffer (0.1% BSA in
PBS). Next, the cells were treated with anti-mouse IgG conjugated with Alexa Fluor 488(1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA). The data were analyzed using the EC800 Cell Analyzer and EC800 software ver. 1.3.6 (Sony Corp.), or the SA3800
Cell Analyzer and SA3800 software ver. 2.05 (Sony Corp.).
2.6. Western Blot Analysis
Cell lysates were prepared using NP-40 lysis buffer (20 mM Tris-HCl [pH 7.5], 150
mM NaCl, 1% Nonidet P-40, and 50 μg/mL of aprotinin) and were denatured in sodium
dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). The 10 μg of proteins were
subjected to electrophoresis using polyacrylamide gels (5–20%; FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan) and transferred onto polyvinylidene difluoride(PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membranes were blockedwith 4% skim milk (Nacalai Tesque, Inc.) in PBST, and were incubated with 10 μg/mL of
C44Mab-94, 10 μg/mL of C44Mab-46, or 0.5 μg/mL of an anti-β-actin mAb (clone AC-15;
Sigma-Aldrich Corp.). Next, the membranes were incubated with anti-mouse immunoglobulins conjugated with peroxidase (diluted 1:1000; Agilent Technologies, Inc.). The
chemiluminescence was performed with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) and detected using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
2.7. Immunohistochemical Analysis
Formalin-fixed paraffin-embedded (FFPE) tissue arrays of gastric carcinoma(BS01012e and BS01011b) and OSCC (OR601c) were obtained from US Biomax Inc. (Rockville, MD, USA). The tissue arrays were autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min. The blocking was performed using SuperBlock
T20 (Thermo Fisher Scientific, Inc.). The sections were incubated with C44Mab-94 (5 μg/mL)
and C44Mab-46 (5 μg/mL), and then treated with the EnVision+ Kit for mouse (Agilent Technologies Inc.). The chromogenic reaction was performed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.). Hematoxylin (FUJIFILM Wako Pure Chemical
Corporation) was used for counterstaining. A Leica DMD108 (Leica Microsystems GmbH,
Wetzlar, Germany) was used to obtain images and examine the sections.
3. Results
3.1. Establishment of an Anti-CD44v8 mAb, C44Mab-94
We previously used CHO/CD44v3–10 cells as an immunogen and generated antiCD44 mAbs, including C44Mab-5 (pan-CD44), C44Mab-3 (v5) [35], C44Mab-9 (v6) [36], andC44Mab-1 (v9) [38]. In this study, we established a new stable transfectant (PANC1/CD44v3–10 cells) as another immunogen (Figure 1A). Mice were immunized with
PANC-1/CD44v3–10 cells (Figure 1B), and hybridomas were seeded in 96-well plates (Figure 1C). The supernatants, which are positive for CHO/CD44v3–10 cells and negative for
CHO-K1 cells, were selected using flow cytometry (Figure 1D). After cloning by limiting
dilution, anti-CD44-mAb-producing clones were finally established. Among establishedclones, we focused on C44Mab-94 (IgG1, kappa), and the epitopes were determined by flow
cytometry and/or ELISA (Figure 1E).

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Figure 1. Anti-human CD44 mAbs production. (A) The structure of CD44s and CD44v. The mRNAof CD44s is assembled by the constant exons (1–5) and (16–20), and produces the standard isoform,
CD44s. The mRNAs of CD44v are generated by the alternative splicing of variant exons. CD44v3–
10 is an immunogen. CD44v3, 8–10, CD44v6–10, CD44v8–10, and CD44v3, 8 are detected in GC cellline [39]. (B) PANC-1/CD44v3–10 was used as an immunogen. (C) The hybridomas were produced
by fusion with splenocytes and P3U1 cells. (D) The screening was performed using parental CHOK1 and CHO/CD44v3–10 cells by flow cytometry. (E) A clone C44Mab-94 was established. Furthermore, the binding epitope was determined by flow cytometry using CD44 deletion mutant-expressed CHO-K1 cells and ELISA.

To determine the C44Mab-94 epitope, we examined the reactivity to CHO/CD44v3–
10 and the N-terminal CD44v3–10 deletion mutants (dN224, dN266, dN304, dN343,
dN386, dN430, dN464, dN494, and dN562)-expressed CHO-K1 cells by flow cytometry(Figure 2A). As shown in Figure 2B, C44Mab-94 reacted with dN224, dN266, dN304,
dN343, dN386, dN430, and CD44v3–10. In contrast, the reactivity completely disappearedin dN464, dN494, and dN562. Because CD44v3–10 and the deletion mutants possess PA16

Antibodies 2023, 12, 45

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tag at the N-terminus, we could confirm all expression on the cell surface by an anti-PA16
tag mAb, NZ-1 (Figure 2C). These results suggest that C44Mab-94 recognizes the v8-encoding sequence.

Figure 2. Epitope determination of C44Mab-94 using deletion mutants of CD44v3–10. (A) The
CD44v3–10 deletion mutants expressed on CHO-K1 cells. (B) The CD44v3–10 mutants-expressed
CHO-K1 cells were incubated with C44Mab-94 (B, red line) or an anti-PA tag mAb, NZ-1 (C, redline), or control blocking buffer (black line), followed by secondary antibodies treatment. The data
were analyzed using the EC800 Cell Analyzer.

To further assess the C44Mab-94 epitope, we performed ELISA using synthetic peptides from the v7- to v9-encoded sequences. As shown in Figure 3, C44Mab-94 reacted with

Antibodies 2023, 12, 45

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CD44p431–450 (DSSHSTTLQPTANPNTGLVE, v8 region), but not with another v8 region(CD44p441–460), or v7/v8 region (CD44p421–440), or v8/v9 region (CD44p451–470). These
results indicated that C44Mab-94 recognizes the CD44 variant-8-encoded sequence, but not
the border sequence between v7 and v8, or between v8 and v9.

Figure 3. Determination of C44Mab-94 epitope by ELISA. Four synthesized peptides, which cover
the CD44v7 to v9 region, were immobilized on immunoplates. The plates were incubated withC44Mab-94 or buffer control (–1st Ab), followed by incubation with peroxidase-conjugated anti-mouse
immunoglobulins. Optical density was measured at 655 nm. ELISA—enzyme-linked immunosorbent
assay. NC—negative control (solvent; DMSO in PBS). Error bars represent means ± SDs.

3.2. Flow Cytometric Analysis of C44Mab-94 against CD44-Expressing Cells
We next examined the reactivity of C44Mab-94 against CHO/CD44v3–10 and
CHO/CD44s cells by flow cytometry. C44Mab-94 recognized CHO/CD44v3–10 cells in adose-dependent manner (Figure 4A). In contrast, C44Mab-94 recognized neither
CHO/CD44s (Figure 4B) nor CHO-K1 (Figure 4C) cells. We confirmed that a pan-CD44
mAb, C44Mab-46 [30], recognized both CHO/CD44s and CHO/CD44v3–10 cells (Supplementary Figure S1A and B, respectively), but not CHO-K1 cells (Supplementary Figure S1C). Furthermore, C44Mab-94 also recognized the OSCC cell line (HSC-3) and GC cell lines (MKN45,
and NUGC-4) in a dose-dependent manner (Figure 4D, E, and F, respectively).

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Figure 4. Flow cytometric analysis of C44Mab-94. CHO/CD44v3–10 (A), CHO/CD44s (B), CHO-K1(C), HSC-3 (D), MKN45 (E), and NUGC-4 (F) cells were incubated with 0.01–10 μg/mL of C44Mab94 (red line) or control blocking buffer (black line). Next, the cells were treated with anti-mouse IgG
conjugated with Alexa Fluor 488. The data were analyzed using the SA3800 Cell Analyzer.

Antibodies 2023, 12, 45

9 of 173.3. Western Blot Analysis
We next conducted western blot analysis to assess the sensitivity of C44Mab-94. Total
cell lysates from CHO-K1, CHO/CD44s, CHO/CD44v3–10, HSC-3, MKN45, and NUGC-4
were examined. As shown in Figure 5A, C44Mab-94 mainly detected CD44v3–10 at bandsof ~75 kDa and over 180 kDa. Furthermore, C44Mab-94 detected endogenous CD44v8 atbands of over 100 kDa in HSC-3, MKN45, and NUGC-4 cells. An anti-pan-CD44 mAb,
C44Mab-46, recognized the lysates from both CHO/CD44s (~75 kDa) and CHO/CD44v3–
10 (>180 kDa) (Figure 5B). Although C44Mab-46 strongly recognized the lysates from
NUGC-4, the reactivity to the HSC-3 and MKN45 lysates was weak. These results indicated that C44Mab-94 specifically detects exogenous CD44v3–10 and endogenous CD44v8.

Figure 5. Western blot analysis using C44Mab-94. The cell lysates (10 μg) of CHO-K1, CHO/CD44s,
CHO/CD44v3–10, HSC-3, MKN45, and NUGC-4 were electrophoresed and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with 10 μg/mL of C44Mab-94 (A),
10 μg/mL of C44Mab-46 (B), and 0.5 μg/mL of an anti-β-actin mAb (C). Next, the membranes wereincubated with anti-mouse immunoglobulins conjugated with peroxidase. The black arrows indicate CD44s (~75 kDa). The red arrows indicate CD44v3–10 (>180 kDa). CD44v8 was broadly detected
in HSC-3, MKN45, and NUGC-4 lysates. The white arrow indicates a band at ~75 kDa recognizedby C44Mab-94 in the CHO/CD44v3–10 lysate. A colored marker was used in the analysis.

3.4. Immunohistochemical Analysis Using C44Mab-94 against Tumor Tissues
We next investigated whether C44Mab-94 could be applied to immunohistochemical
analysis using FFPE sections. We first examined the reactivity of C44Mab-94 in the OSCC
tissue array because this type was revealed as the second highest CD44-positive cancer
type in the Pan-Cancer Atlas [40]. As shown in Supplementary Figure S2A,B, the membranous staining in OSCC was observed by C44Mab-94 and C44Mab-46. In a stromal-invaded
OSCC section, C44Mab-94 strongly stained invaded OSCC and could clearly distinguishtumor cells from stromal tissues (Supplementary Figure S2C). In contrast, C44Mab-46
stained both invaded OSCC and surrounding stroma cells (Supplementary Figure S2D).
Supplementary Table S1 summarizes the results of OSCC tissue staining.

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We next stained the GC tissue array (BS01011b) using C44Mab-94 and C44Mab-46.
C44Mab-94 exhibited membranous staining in IGC (Figure 6A). C44Mab-46 also stained thesame type of cancer cells (Figure 6B). Furthermore, membranous and cytoplasmic staining
by C44Mab-94 and C44Mab-46 was observed in stromal-invaded tumor cells (Figure 6C,D).
In DGC (Figure 6E,F), diffusely spread tumor cells were strongly stained by both C44Mab94 and C44Mab-46. In contrast, neither C44Mab-94 nor C44Mab-46 stained the ductal epithelial structure of IGC (Figure 6G,H). Additionally, stromal staining by C44Mab-46 was observed in the tissue (Figure 6H).
We summarized the immunohistochemical analysis of GC in Table 1; C44Mab-94
stained 28 out of 72 cases (39%) of GC. A similar staining was also observed in anothertissue array (BS01012e, Supplementary Figure S3). We summarized the data of immunohistochemical analysis in Supplementary Table S2. These results indicated that C44Mab-94
is useful to detect CD44v8 in immunohistochemical analysis of FFPE tumor sections.

Figure 6. Immunohistochemistry using C44Mab-94 and C44Mab-46 against GC. (A–H) The serial sections of GC tissue arrays (BS01011b) were treated with 5 μg/mL of C44Mab-94 or 5 μg/mL of C44Mab46, followed by treatment with the Envision+ kit. The chromogenic reaction was performed using
DAB, and the sections were counterstained with hematoxylin. Scale bar = 100 μm.

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Table 1. Immunohistochemical analysis using C44Mab-94 and C44Mab-46 against GC tissue array(BS01011b).

No. Age Sex
Pathology diagnosis
1 55 F
Adenocarcinoma
2 51 F
Adenocarcinoma
3 71 M
Adenocarcinoma
4 63 M
Adenocarcinoma
5 61 M
Adenocarcinoma
6 61 M
Adenocarcinoma
7 60 M
Adenocarcinoma
8 54 M
Adenocarcinoma
9 46 F
Adenocarcinoma
10 66 M Mucinous adenocarcinoma
11 56 M
Adenocarcinoma
12 52 F
Adenocarcinoma
13 70 M
Adenocarcinoma
14 71 M
Adenocarcinoma
15 61 M
Adenocarcinoma
16 75 M
Adenocarcinoma
17 72 F
Adenocarcinoma
18 60 M
Adenocarcinoma
19 63 F
Adenocarcinoma
20 69 M
Adenocarcinoma
21 54 F
Adenocarcinoma
22 50 F
Adenocarcinoma
23 64 M
Adenocarcinoma
24 59 M
Adenocarcinoma
25 59 M
Adenocarcinoma
26 44 M
Adenocarcinoma
27 76 M
Adenocarcinoma
28 56 M
Adenocarcinoma
29 56 M
Adenocarcinoma
30 58 M
Adenocarcinoma
31 94 M
Adenocarcinoma
32 56 F
Adenocarcinoma
33 56 M
Adenocarcinoma
34 51 F
Adenocarcinoma
35 67 M
Adenocarcinoma
36 53 M
Adenocarcinoma
37 48 F
Adenocarcinoma
38 58 M
Adenocarcinoma
39 61 M
Adenocarcinoma
40 62 M
Adenocarcinoma
41 65 M
Adenocarcinoma
42 47 F
Adenocarcinoma
43 65 M
Adenocarcinoma
44 52 F
Adenocarcinoma
45 72 M
Adenocarcinoma
46 68 F
Adenocarcinoma

TNM Grade Stage C44Mab-94 C44Mab-46
T2N0M01IB+
T2N0M0IB
T3N1M01
IIB++
T3N0M0
1
IIA
T2N0M01IB
T2N0M01IB+
T3N2M01
IIIA
T3N2M0
1
IIIA+++
T3N0M0
1
IIA+
T3N0M0 2–3 IIA+
T2N0M02
IB+++
T3N0M02
IIA++
T3N0M02
IIA+
T2N0M02
IB++
T3N0M02
IIA
T3N1M02
IIB
T3N0M02
IIA++
T3N0M02
IIA
T3N0M02
IIA
T2N0M02
IB
T3N0M02
IIA
T3N0M03IIA
T3N0M03
IIA++
T2N0M02IB+++++
T2N0M02
IB
T3N0M02
IIA
T3N0M02
IIA+
T3N0M02
IIA+
T2N0M02
IB++
T3N0M02
IIA+
T2N0M02IB++
T2N0M03IB++
T4N1M03
IIIA++
T3N0M02
IIA++
T3N0M02
IIA++
T3N0M0 2–3 IIA++
T2N1M03
IIA++++
T2N0M02IB
T2N0M03IB+
T2N0M03IB+
T2N0M03IB++
T3N1M03
IIB
T2N0M0IB
T2N0M03IB
T2N0M03IB+
T3N0M03
IIA++

Antibodies 2023, 12, 45

12 of 17

47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72

56
59
62
60
64
69
75
48
59
64
55
58
64
67
49
35
45
43
56
66
60
74
58
68
50
51

M
Adenocarcinoma
T3N0M0M
Adenocarcinoma
T3N1M0M
Adenocarcinoma
T3N1M0M
Adenocarcinoma
T3N1M0M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T3N0M0M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T3N0M0M
Adenocarcinoma
T3N0M0M
Adenocarcinoma
T3N1M0M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T3N1M0F
Adenocarcinoma
T4N0M1
M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T3N0M0M
Adenocarcinoma
T2N0M0M
Adenocarcinoma
T2N0M0
M Mucinous adenocarcinoma T2N0M0
M Mucinous adenocarcinoma T3N0M0
M Papillary adenocarcinoma T2N0M0

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3
2

IIA
IIB
IIB
IIB
IB
IB
IB
IB
IB
IIA
IB
IIA
IIA
IIB
IB
IIB
IV
IB
IB
IB
IIA
IB
IB
IB
IIA
IB

++
+
+
+
+
+
+
++
+
+
+
+
+
+
-

++
+
+++
+
++
++
+
++
+
+
+
++
+
+
++
+
+

-, no stain; +, weak intensity; ++, moderate intensity; +++, strong intensity.

4. Discussion
The VFF series anti-CD44v mAbs were previously established by the immunizationof bacterial-expressed CD44v3–10 and glutathione S-transferase fusion protein [41,42].
The clones, VFF-8 (v5), VFF-18 (v6), VFF-9 (v7), VFF-17 (v7/8), and VFF-14 (v10) have beenused for various applications [43–45]. Furthermore, VFF-18 was humanized as BIWA-4[46], and developed to bivatuzumab-mertansine, an antibody-drug conjugate (ADC), forclinical trials [47,48]. An anti-CD44v3 mAb (clone 3G5) [49] and an anti-CD44v9 mAb(clone RV3) [26] were also developed and widely used for research studies. However, aCD44v8-specific mAb had not been developed.
In this study, we developed a novel anti-CD44v8 mAb—C44Mab-94—using the CBIS
method (Figure 1). We determined the epitope as a v8-encoded region using deletion mutants of CD44 (Figure 2), and synthetic peptides (Figure 3). We have established anti-CD44
mAbs using CHO/CD44v3–10 [29,35,36,38], PANC-1/CD44v3–10 (in this study), or purified CD44v3–10 ectodomain [30,37] as antigens. We listed them in our original “Antibody
Bank” (see Supplementary Materials). However, clones which recognize the v8-encoded
region were rare, suggesting that the region has low antigenicity and/or locates the insideof CD44v3–10 protein. Although the affinity of C44Mab-94 is low against target cells,
C44Mab-94 can be applied to various applications, including flow cytometry (Figure 4),
western blotting (Figure 5), and immunohistochemistry (Figure 6).
We confirmed that C44Mab-94 recognizes a synthetic peptide of the v8-encoded region (DSSHSTTLQPTANPNTGLVE), but not border regions (v7/v8 and v8/v9) by ELISA(Figures 3). The epitope region possesses multiple confirmed and predicted O-glycosylation sites [50]. C44Mab-94 recognized a ~75-kDa band in the lysate of CHO/CD44v3–10

Antibodies 2023, 12, 45

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(Figure 5A), which is similar to the predicted molecular size from the amino acids ofCD44v3–10. Therefore, C44Mab-94 could recognize CD44v3–10 regardless of the glycosylation. A detailed epitope analysis and an investigation of the influence of glycosylation on
C44Mab-94 recognition are required in future studies.
In a GC cell line, the major transcripts of CD44v, including CD44v3, 8–10, CD44v6–
10, CD44v8–10, and CD44v3, 8 were identified [39] (Figure 1A). C44Mab-94 can cover all
products of the transcripts and detect the broad CD44v-expressing GC. Since CD44v8–10
plays critical roles in the regulation of ROS defense and GC progression [26], an antiCD44v9 mAb (clone RV3) has so far mainly been used in immunohistochemistry. Several
studies revealed that CD44v9 is a predictive marker for the recurrence of GC [51] and abiomarker for GC patient selection and efficacy of the xCT inhibitor, sulfasalazine [52]. Further
investigations are required to reveal the relationship between CD44v8 expression and clinicalfactors using C44Mab-94. Additionally, C44Mab-94 recognized both IGC (Figure 6A) and DGC(Figure 6E) in immunohistochemistry. It would be worthwhile investigating whether CD44v8
is expressed in a specific molecular subtype of GC [6] in a future study.
A comprehensive analysis of malignant ascites identified the amplifications of cancer
driver genes including CD44 [10]. Although the expression pattern of CD44v is not identified, CD44v8 is thought to be an important target for mAb therapy due to the commonly
included region in GC [39]. We have shown the antitumor activity using class-switchedand defucosylated IgG2a recombinant mAbs [33]. The defucosylated IgG2a mAbs were produced by CHO-K1 lacking fucosyltransferases 8; they exhibited potent ADCC activity invitro, and suppressed the growth of xenograft [53]. Therefore, the production of defucosylated C44Mab-94 is one of the strategies used to evaluate the antitumor effect on GC withperitoneal metastasis in the preclinical model.
Clinical applications of a humanized anti-CD44v6 mAb (BIWA-4) bivatuzumabmertansine drug conjugate to solid tumors failed because of skin toxicities [47,48]. The
accumulation of the mertansine drug was thought to be a cause of the toxicity [47,48].
Human acute myeloid leukemia (AML) cells also express high levels of CD44 mRNA due
to the suppression of methylation of the CpG islands in the promoter [54]. Furthermore,higher expression of CD44v6 was observed in AML patients with FLT3 or DNMT3A mutations. Therefore, a mutated version of BIWA-4—called BIWA-8—was engineered to develop chimeric antigen receptors (CARs) for AML. The CD44v6 CAR-T cells exhibited potent anti-leukemic effects [54,55] indicating that CD44v6 is a rational target of CAR-T therapy for AML harboring FLT3 or DNMT3A mutations. Additionally, the CD44v6 CAR-T
also showed an antitumor effect in lung and ovarian cancer xenograft models [56], whichis expected for a wider development toward solid tumors.
Since CD44 mRNA is elevated in AML, other CD44 variants might also be transcribedin AML. Furthermore, CD44v8–10 was elevated during chronic myeloid leukemia (CML)
progression from chronic phase to blast crisis in a humanized mouse model, which is required for the maintenance of stemness of CML [57]. Therefore, in future, we will investigate the reactivity of C44Mab-94 against hematopoietic malignancy. Further studies are
required to investigate the selective expression of CD44v8 in leukemia cells, but not inhematopoietic stem cells, to certify its safety as a CAR-T antigen.
In this study, we used tumor cell-expressed CD44v3–10 as an immunogen. This strategy is important for the establishment of cancer-specific mAbs (CasMabs). We previously
developed PDPN-targeting CasMabs [58] and podocalyxin-targeting CasMabs [59], which
recognize cancer-type aberrant glycosylation of the targets [60]. Anti-PDPN-CasMabs are
currently applied to CAR-T therapy in preclinical models [61]. For CasMab development,
we should do further screening of our established anti-CD44 mAbs by comparing the reactivity against normal cells. Anti-CD44 CasMabs could be applicable for designing themodalities including ADCs and CAR-T.

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Supplementary Materials: The following supporting information can be downloaded at:https://www.mdpi.com/article/10.3390/antib12030045/s1, Figure S1: Recognition of CHO/CD44s,
CHO/CD44v3–10, and cancer cell lines by C44Mab-46 using flow cytometry, Figure S2: Immunohistochemistry using C44Mab-94 and C44Mab-46 against OSCC, Figure S3: Immunohistochemistry
using C44Mab-94 and C44Mab-46 against GC, Table S1: Immunohistochemical analysis using C44Mab94 and C44Mab-46 against OSCC tissue array, and Table S2: Immunohistochemical analysis using
C44Mab-94 and C44Mab-46 against GC tissue array (BS01012e). We listed the information on antiCD44 mAbs in our original “Antibody Bank” (http://www.med-tohoku-antibody.com/topics/001_paper_antibody_PDIS.htm#CD44 (accessed on 27 June 2023)).
Author Contributions: N.G., H.S., T.T. and T.O. performed the experiments. M.K.K. and Y.K. designed the experiments. N.G., H.S. and Y.K. analyzed the data. N.G., H.S. and Y.K. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported in part by the Japan Agency for Medical Research and Development (AMED) under grant nos. JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.), JP22bm1004001 (toY.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.); and in part by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) under grant nos. 21K20789(to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Institutional Review Board Statement: The animal study protocol was approved by the Animal Careand Use Committee of Tohoku University (Permit number: 2019NiA-001) for studies involving animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article and Supplementary Materials.
Conflicts of Interest: The authors declare no conflict of interest.

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