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Communication

Establishment of a Highly-Sensitive Anti-EphB2
Monoclonal Antibody Eb2Mab-3 for Flow Cytometry
Rena Ubukata, Hiroyuki Suzuki, Miu Hirose, Hiroyuki Satofuka, Tomohiro Tanaka,
Mika K. Kaneko and Yukinari Kato *Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai, Miyagi 980-8575, Japan; ubukata.rena.p7@dc.tohoku.ac.jp (R.U.);
hiroyuki.suzuki.b4@tohoku.ac.jp (H.Suzuki.); hirose.miu.r4@dc.tohoku.ac.jp (M.H.);
hiroyuki.satofuka.e1@tohoku.ac.jp (H.Satofuka.); tomohiro.tanaka.b5@tohoku.ac.jp (T.T.);mika.kaneko.d4@tohoku.ac.jp (M.K.K.)* Correspondence: yukinari.kato.e6@tohoku.ac.jp; Tel.: +81-22-717-8207

Abstract: EphB2 is a member of the Eph family tyrosine kinase receptors. EphB2 binds to ephrinB1, ephrin-B2, and ephrin-B3, which are critical regulators of vascular and nervous development
through controlling cell migration and axon guidance. EphB2 is overexpressed in tumors, including
glioma, breast cancer, hepatocellular carcinoma, and malignant mesothelioma, and it functions as atumor promoter. Therefore, the development of monoclonal antibodies (mAbs) against EphB2 isessential for tumor diagnosis and therapy for EphB2-positive tumors. In this study, we developed
novel mAbs for human EphB2 using the Cell-Based Immunization and Screening (CBIS) method.
One of the established anti-EphB2 mAbs, Eb2Mab-3 (mouse IgG1, kappa), reacted with EphB2overexpressed Chinese hamster ovary-K1 (CHO/EphB2) and an endogenously EphB2-expressing
cancer cell line (LS174T) by flow cytometry. Using flow cytometry, the dissociation constant (KD)values of Eb2Mab-3 for CHO/EphB2 and LS174T were determined as 1.1 × 10−9 M and 3.6 × 10−10 M,
respectively. These results indicated that Eb2Mab-3 possesses a high affinity for detecting EphB2
and could apply to tumor therapy.
Keywords: EphB2; monoclonal antibody; Cell-Based Immunization and Screening; flow cytometry

1. Introduction
The mammalian ephrin and Eph system includes eight cell surface ephrin ligands (five ephrinAs and three ephrin-Bs) and 14 receptor tyrosine kinases (nine EphA and five EphB receptors) [1].
Eph receptors make the complex to ephrins with dimerization or oligomerization, which leads to thetyrosine phosphorylation of Eph receptor and ephrin-B [2]. The phosphorylated tyrosine recruits
cytoplasmic effectors containing src-homology 2 (SH2) domains, phosphotyrosine-binding (PTB)domains, and PDZ domains [3]. Therefore, the Eph receptor and ephrin complexes activate
bidirectional (forward and reverse, respectively) signaling, which is essential for communication in
the same or different types of cells [4]. Through bidirectional signaling, the Eph system regulates
tissue development, homeostasis, and regeneration; the dysregulation causes many diseases
including cancer [1].
The dysregulation of the Eph system is observed in both tumor cells and tumor
microenvironment [5]. The Eph system plays distinct roles in tumor development and functions asboth tumor promoters and suppressors in a cellular context-dependent manner [5]. EphB2 isoverexpressed in several tumors, such as glioblastoma [6], breast cancer [7], hepatocellular carcinoma[8], and malignant mesothelioma [9], which is associated with poor clinical outcomes. In these tumors,
EphB2 promotes the migration and invasion via forward signaling [10,11].
In contrast, the expression of EphB2 is downregulated in some tumors such as colorectal cancer[12]. In the intestinal epithelium, EphB receptors promote stem and progenitor proliferation [13]. The© 2024 by the author(s). Distributed under a Creative Commons CC BY license.

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intestinal epithelial cell migration is deranged in mice lacking EphB2 and EphB3, and the absence of
EphB signaling results in ~50% reduction in the number of proliferating cells [13]. Furthermore, EphB
receptor expression is elevated in intestinal adenomas [14]. In contrast, EphB2 function as tumor
suppressors by inhibiting the invasive growth. EphB signaling promotes adherens junction formationof colorectal cancer cells, suppressing cancer progression by inhibiting invasive growth [15]. EphB2
expression is lost during progression to carcinoma and initiation of invasive growth [16].
To evaluate the expression of EphB2 and targeting the EphB2-positive cancer cells, thedevelopment of monoclonal antibodies (mAbs) against EphB2 is essential. We have developed antireceptor tyrosine kinase mAbs against human epidermal growth factor (EGFR) (clone EMab-17) [17],mouse EGFR (EMab-300) [18], HER2 (H2Mab-19) [19], mouse HER2 (H2Mab-304) [20], and HER3
(H3Mab-17) [21] using the Cell-Based Immunization and Screening (CBIS) method. The CBIS method
includes immunizing antigen-overexpressed cells and high-throughput hybridoma screening using
flow cytometry. In this study, novel anti-EphB2 mAbs were developed by the CBIS method.
2. Materials and Methods
2.1. Antibodies
OptiBuild™ RB545 mouse anti-human EphB2 mAb (clone 2H9; mouse IgG1, kappa) was
purchased from BD Bioscience (Franklin Lakes, NJ). Alexa Fluor 488-conjugated anti-mouse IgG waspurchased from Cell Signaling Technology, Inc. (Danvers, MA).
2.2. Preparation of Cell Lines
LS174T (human colorectal cancer), LN229 (human glioblastoma), Chinese hamster ovary (CHO)K1, and P3X63Ag8U.1 (P3U1) were obtained from the American Type Culture Collection (Manassas,
VA).
pCMV6-myc-DDK vector with EphB2 (Catalog No.: RC223882, Accession No.: NM_004442) was
purchased from OriGene Technologies, Inc. (Rockville, MD). The EphB2 plasmids were transfected
into CHO-K1 and LN229 cells using a Neon transfection system (Thermo Fisher Scientific Inc.,
Waltham, MA). Stable transfectants were established through cell sorting using the RB545-conjugatedanti-human EphB2 (2H9) mAb and a cell sorter (SH800; Sony Corp., Tokyo, Japan), after which
cultivation in a medium containing 0.5 mg/mL of G418 (Nacalai Tesque, Inc., Kyoto, Japan) was
performed.
CHO-K1, EphB2-overexpressed CHO-K1 (CHO/EphB2), and P3U1 were cultured in a Roswell
Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), with 10% heatinactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc.), 100 units/mL of penicillin, 100
μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B (Nacalai Tesque, Inc.). LS174T, LN229,
and EphB2-overexpressed LN229 (LN229/EphB2) were cultured in Dulbecco’s Modified Eagle
Medium (DMEM) (Nacalai Tesque, Inc.), supplemented with 10% FBS, 100 units /mL of penicillin,
100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. All cells were grown in a humidified
incubator at 37°C in an atmosphere of 5% CO2 and 95% air.
2.3. Development of Hybridomas
The animal was housed under specific pathogen-free conditions. All animal experiments wereapproved by the Animal Care and Use Committee of Tohoku University (Permit number: 2022MdA001).
Two five-week-old BALB/cAJcl mice were purchased from CLEA Japan (Tokyo, Japan). To
develop mAbs against EphB2, we intraperitoneally immunized the mice with LN229/EphB2 (1 × 108
cells) plus Alhydrogel adjuvant 2% (InvivoGen). The procedure included three additional weekly
injections (1 × 108 cells/mouse), followed by a final booster intraperitoneal injection (1 × 108
cells/mouse) two days before harvesting spleen cells. The harvested spleen cells were subsequently
fused with P3U1 cells, using PEG1500 (Roche Diagnostics, Indianapolis, IN), after which hybridomas
were grown in the RPMI-1640 medium with 10% FBS, 5% Briclone (NICB, Dublin, Ireland), 100

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units/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B. For the
hybridoma selection, hypoxanthine, aminopterin, and thymidine (HAT; Thermo Fisher ScientificInc.) were added to the medium. The supernatants were subsequently screened using flow cytometry
using CHO/EphB2 and CHO-K1.
The cultured supernatant of Eb2Mab hybridomas was applied to 1 mL of Ab-Capcher(ProteNova, Kagawa, Japan). After washing with phosphate-buffered saline (PBS), the antibodies
were eluted with an IgG elution buffer (Thermo Fisher Scientific Inc.). Finally, the eluates wereconcentrated, and the elution buffer was replaced with PBS using Amicon Ultra (Merck KGaA,
Darmstadt, Germany).
2.4. Flow Cytometric Analysis
Cells were harvested after brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic
acid (EDTA, Nacalai Tesque, Inc.). The cells were washed with 0.1% bovine serum albumin (BSA) in
PBS (blocking buffer) and treated with 0.01, 0.1, 1, and 10 μg/mL of primary mAbs for 30 min at 4°C.
The cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2,000). The cells were also
suspended in 0.01, 0.1, 1, and 10 μg/mL of the RB545-conjugated anti-human EphB2 mAb (2H9). The
fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corp), and the data wereanalyzed using FlowJo (BD Biosciences).
2.5. Determination of Dissociation Constant (KD) by Flow Cytometry
CHO/EphB2 and LS174T cells were suspended in serially-diluted Eb2Mabs for 30 min at 4°C.
The cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:200). The cells were also
suspended in serially-diluted RB545-conjugated anti-human EphB2 mAb (2H9). The fluorescencedata were collected using a FACSLyric (BD Biosciences). The KD was calculated by fitting saturation
binding curves to the built-in, one-site binding models in GraphPad PRISM 6 (GraphPad Software,Inc., La Jolla, CA).
3. Results
3.1. Development of Anti-EphB2 mAbs Using the CBIS Method
To develop anti-EphB2 mAb, mice were immunized with LN229/EphB2 cells (Figure 1A). The
spleen was then excised from the mice, and splenocytes were fused with myeloma P3U1 cells (Figure
1B). The developed hybridomas were subsequently seeded into ten 96-well plates and cultivated forsix days. The positive wells were screened by selecting CHO/EphB2-reactive and CHO-K1-nonreactive supernatants using flow cytometry (Figure 1C). We finally obtained 133 positive wells(13.9%) out of 956 wells. After the limiting dilution of the part of positive wells and several additional
screenings, twelve clones were finally established (Figure 1D, Supplementary Table S1).

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Figure 1. The production of anti-EphB2 mAbs (A) LN229/EphB2 cells were immunized into two
BALB/cAJcl mice. (B) The spleen cells were fused with P3U1 cells. (C) To select anti-EphB2 mAbproducing hybridomas, the supernatants were screened by flow cytometry using CHO-K1 andCHO/EphB2 cells. (D) After limiting dilution, anti-EphB2 mAbs were finally established.

3.2. Flow Cytometric Analysis Using Anti-EphB2 mAbs
We next focus on eight mouse IgG1 clones (Eb2Mab-1, 2, 3, 4, 7, 8, 10, and 12) and purified themAbs from supernatants (Supplementary Table S1). We conducted flow cytometry using theEb2Mabs and 2H9 (from BD Biosciences) against CHO/EphB2, CHO-K1, LN229/EphB2, and LN229
cells. Eb2Mabs recognized CHO/EphB2 cells dose-dependently at 10, 1, 0.1, and 0.01 μg/mL(Supplementary Figure S1). Among Eb2Mabs, Eb2Mab-3 showed the highest reactivity (Figure 2A).
2H9 also recognized CHO/EphB2 cells dose-dependently at 10, 1, 0.1, and 0.01 μg/mL, which are lesseffective compared to Eb2Mab-3 (Figure 2A). Parental CHO-K1 cells were not recognized even at 10
μg/mL of Eb2Mab-3 and 2H9 (Figure 2B). The superior reactivity of Eb2Mab-3 compared to 2H9 was
also observed in LN229/EphB2 and LN229 cells (Supplementary Figure S2). The weak expression ofendogenous EphB2 in LN229 was previously confirmed by quantitative PCR and western blot
analyses [22].

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Figure 2. Flow cytometry of EphB2-expressed CHO-K1 cells using Eb2Mab-3 and 2H9. CHO/EphB2(A) and CHO-K1 (B) cells were treated with 0.01–10 μg/mL of Eb2Mab-3 or 2H9 conjugated with
RB545 (Red line). The Eb2Mab-3 treated cells were further incubated with anti-mouse IgG conjugatedwith Alexa Fluor 488. The fluorescence data were subsequently collected using the SA3800 Cell
Analyzer. The black line represents the negative control (blocking buffer).

We next investigated the reactivity of Eb2Mabs and 2H9 against an endogenous EphB2expressing colorectal cancer cell line, LS174T [23]. Eb2Mabs recognized LS174T cells dosedependently at 10, 1, 0.1, and 0.01 μg/mL (Supplementary Figure S3). Among Eb2Mabs, Eb2Mab-3
also showed the highest reactivity (Figure 3). In contrast, 2H9 could react with LS174T cells at morethan 0.1 μg/mL (Figure 3). These results suggest that Eb2Mab-3 specifically recognizes EphB2, and isalso helpful in detecting endogenous EphB2 by flow cytometry.

Figure 3. Flow cytometry of endogenous EphB2-expressing cells using Eb2Mab-3 and 2H9. LS174T
cells were treated with 0.01–10 μg/mL of Eb2Mab-3 or 2H9 conjugated with RB545 (Red line). The
Eb2Mab-3 treated cells were further incubated with anti-mouse IgG conjugated with Alexa Fluor 488.
The fluorescence data were subsequently collected using the SA3800 Cell Analyzer. The black line
represents the negative control (blocking buffer).

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3.3. Determination of the Binding Affinity of Eb2Mabs and 2H9 Using Flow Cytometry
To determine the KD values of Eb2Mabs and 2H9, we conducted flow cytometry, and thegeometric mean of the fluorescence intensity was plotted versus the concentration of mAbs. The KD
values of Eb2Mab-3 and 2H9 for CHO/EphB2 were determined as 1.1 × 10−9 M and 3.4 × 10−9 M,
respectively (Figure 4A). Although we also determined the KD values of other Eb2Mabs, Eb2Mab-3
exhibited the highest affinity (Supplementary Table S1). We next determined the KD values of
Eb2Mab-3 and 2H9 for LS174T as 3.6 × 10−10 M and 1.9 × 10−9 M, respectively (Figure 4B). These results
indicate that Eb2Mab-3 possesses the superior affinity to CHO/EphB2 and LS174T compared to thatof 2H9.

Figure 4. The binding affinity of Eb2Mab-3 and 2H9. CHO/EphB2 (A) and LS174T (B) cells weresuspended in serially diluted Eb2Mab-3. The cells were treated with anti-mouse IgG conjugated with
Alexa Fluor 488. The cells were also suspended in serially diluted 2H9 conjugated with RB545. The
fluorescence data were subsequently collected using the FACSLyric, followed by the calculation of
the KD using GraphPad PRISM 6.

4. Discussion
An anti-EphB2 mAb, clone 2H9, was extensively characterized and developed for tumor therapy
as an antibody-drug conjugate [24], The 2H9 was established by the immunization of mice with the
EphB2 ectodomain produced by baculovirus expression system [24]. In this study, we establishedanti-EphB2 mAbs using the CBIS method (Figure 1). Among the established mAbs, Eb2Mab-3
exhibited the superior reactivity compared to 2H9 in CHO/EphB2 (Figure 2), LN229/EphB2(Supplemental Figure S2), and LS174T (Figure 3) cells. Furthermore, Eb2Mab-3 possesses a higher
affinity to CHO/EphB2 and LS174T than that of 2H9 (Figure 4).
The 2H9 effectively blocked the interaction of EphB2 with the ligands and inhibited the
autophosphorylation of EphB2 [24]. However, 2H9 did not affect the proliferation of EphB2-positive
tumor cells [24]. The identification of the epitope is essential to assess the properties of Eb2Mab-3 andPreprints.org (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 June 2024

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2H9. We have developed the RIEDL insertion for epitope mapping (REMAP) and PA insertion for
epitope mapping (PAMAP) methods to determine the conformational epitopes of mAbs. The
epitopes of anti-EGFR mAbs (EMab-51 and EMab-134) [25,26] and anti-CD44 mAbs (C44Mab-5 andC44Mab-46) [27,28] could be determined using the REMAP method. Furthermore, the epitopes of antimouse CD39 mAb (C39Mab-1) could be determined using both REMAP and PAMAP methods [29].
Therefore, further studies are required to determine the epitope and biological activities of Eb2Mab3.
We investigated the expression of EphB2 in more than 100 cell lines using flow cytometry.
LS174T exhibited the highest expression (Figure 3). Since LS174T is a transplantable cancer cell line
in BALB/c nude mice [30], in vivo antitumor effects of Eb2Mab-3 could be evaluated. To perform this,converting Eb2Mab-3 (mouse IgG1) to mouse IgG2a subclass is essential for enhancing effector
activation ability. We previously produced recombinant mAbs, which were converted into the mouse
IgG2a subclass from mouse IgG1. Furthermore, we produced defucosylated IgG2a mAbs using
fucosyltransferase 8-deficient CHO-K1 cells to enhance the antibody-dependent cellular cytotoxicity
and in vivo antitumor effect in mouse xenograft models [31–33]. Therefore, a class-switched anddefucosylated type of Eb2Mab-3 could contribute to the treatment of EphB2-positive tumors in
preclinical studies.
We have developed cancer-specific mAbs (CasMabs) against HER2 (H2Mab-214 and H2Mab-250)[34,35], podocalyxin (PcMab-6 and PcMab-60) [36,37], and podoplanin (LpMab-2 and LpMab-23)[38,39] and revealed that these mAbs react with cancer cells, but not normal cells in flow cytometry.
The strategy used in this study is also applicable to select anti-EphB2 CasMabs from Eb2Mabs(Supplementary Table S1) or clones from remaining positive wells. We have confirmed that EphB2 isdetected in some normal epithelial cells and will screen the anti-EphB2 CasMabs. The uniqueproperty of H2Mab-250 could contribute to developing HER2-targeting chimeric antigen receptor(CAR)-T cells (now in a clinical phase I study in the US). Therefore, developing Eb2Mabs for CAR-T
would be necessary for treating EphB2-positive tumors.
Supplementary Materials: The following supporting information can be downloaded at the website of thispaper posted on Preprints.org. Figure S1: Flow cytometry of EphB2-expressed CHO-K1 cells using Eb2Mabs and2H9.; Figure S2: Flow cytometry of EphB2-expressed LN229 cells using Eb2Mab-3 and 2H9.; Figure S3: Flowcytometry of endogenous EphB2-expressing cells using Eb2Mabs and 2H9.; Table S1: Determination of KD of
Eb2Mabs (IgG1 isotype) using flow cytometry.
Author Contributions: R.U., H.Suzuki., M.H., H.Satofuka, and T.T. performed the experiments. M.K.K. and Y.K.
designed the experiments. R.U. and H.Suzuki. analyzed the data. H.Suzuki. and Y.K. wrote the manuscript. All
authors have read and agreed to the published version of the manuscript.
Funding Information: This research was supported in part by Japan Agency for Medical Research andDevelopment (AMED) under Grant Numbers: JP23ama121008 (to Y.K.), 23bm1123027 (to Y.K.), andJP23ck0106730 (to Y.K.), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific
Research (KAKENHI) grant nos. 22K06995 (to H.S.) and 22K07224 (to Y.K.).
Institutional Review Board Statement: The animal study protocol was approved by the Animal Care and Use
Committee of Tohoku University (Permit number: 2022MdA-001) for studies involving animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: All related data and methods are presented in this paper. Additional inquiries
should be addressed to the corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest involving this article.

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