MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 5, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0015
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Development of an Anti-EphB4 Monoclonal Antibody
for Multiple Applications Against Breast Cancers

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Ren Nanamiya, Hiroyuki Suzuki, Mika K. Kaneko, and Yukinari Kato

The erythropoietin-producing hepatocellular carcinoma (Eph) receptors are the largest receptor tyrosine kinase
family. EphB4 is essential for cell adhesion and motility during embryogenesis. Pathologically, EphB4 isoverexpressed and contributes to poor prognosis in various tumors. Therefore, specific monoclonal antibodies(mAbs) should be developed to predict the prognosis for multiple tumors with high EphB4 expression, including breast and gastric cancers. This study aimed to develop specific anti-EphB4 mAbs for multiple applications using the Cell-Based Immunization and Screening method. EphB4-overexpressed Chinese hamster
ovary (CHO)-K1 (CHO/EphB4) cells were immunized into mice, and we established an anti-EphB4 mAb(clone B4Mab-7), which is applicable for flow cytometry, Western blot, and immunohistochemistry (IHC).
B4Mab-7 reacted with endogenous EphB4-positive breast cancer cell line, MCF-7, but did not react with
EphB4-knockout MCF-7 (BINDS-52) in flow cytometry. Dissociation constant (KD) values were determined tobe 2.9 · 10-9 M and 1.3 · 10-9 M by flow cytometric analysis for CHO/EphB4 and MCF-7 cells, respectively.
B4Mab-7 detected the EphB4 protein bands from breast cancer cells in Western blot, and stained breast cancer
tissues in IHC. Altogether, B4Mab-7 is very useful for detecting EphB4 in various applications.
Keywords: EphB4, monoclonal antibody, Cell-Based Immunization and Screening, immunohistochemistry

Introduction

Breast cancer is a malignant tumor with overwhelmingly high incidence in women. In 2020, there were
estimated 2,261,419 new breast cancer cases (11.7% of allcancer cases) and estimated 684,996 deaths (6.9% of allcancer deaths) worldwide.1 In most developed countries,
breast cancer fatality rates have been declining in recent
years, owing to improved treatment strategies and earlier
detection techniques.2–4 Breast cancer treatment currently
includes surgery, radiation therapy, chemotherapy, and molecular targeted therapy.5–7
Molecular targeted therapy depends on the subtype ofbreast cancer. Breast cancers are categorized into clinicopathological surrogate intrinsic subtypes defined by immunohistochemistry (IHC) testing of estrogen receptor (ER),progesterone receptor (PR), human epidermal growth factor
receptor 2 (HER2), and Ki-67 status.8–10 Breast cancer subtypes can be largely classified into hormone receptor (ER or

PR)-positive luminal A, luminal B, HER2-positive, andtriple-negative breast cancer (TNBC).8–10
The erythropoietin-producing hepatocellular carcinoma(Eph) receptors belong to the largest family of receptor tyrosine kinases in mammals.11 Eph receptors are physiologicallyassociated with angiogenesis, cell migration, vascularization,axon guidance, and neurogenesis.12 The Eph family was first
discovered in human cancer cell lines in 1987,13 and is now
known to have two classes of receptors consisting of nine
EphA members and five EphB members in humans.14
The extracellular region of the Eph receptor consists of aligand binding domain, followed by a cysteine-rich domain(including the sushi and epidermal growth factor-like domains) and two fibronectin domains.15,16 The intracellularside of the Eph receptor consists of a transmembrane region, atyrosine kinase domain, a sterile alpha motif, and a PDZ
domain. The EphA receptor binds promiscuously to ephrin A
ligands (five types) and the EphB receptor binds promiscuously to ephrin B ligands (three types), but there may be

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

166

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DEVELOPMENT OF ANTI-EPHB4 MAB

cross-talk between the groups.17–23 In contrast to other Ephreceptors, EphB4 shows clear specificity for the ligand
ephrinB2 and very weak binding affinity for ephrinB1 andephrinB3.16
EphB4 and ephrinB2 are biologically essential for celladhesion, cell migration, stabilization, branching, and remodeling during embryogenesis for cardiovascular formation.24–30 Mice lacking either EphB4 (expressed in veins) or
ephrinB2 (expressed in arteries) showed identical defects inthe formation of capillary connections between the networkof arteries and veins in the head and yolk sac, resulting inembryonic lethal.29–32 EphB4 and ephrinB2 create a repulsive effect between them and act to prevent the fusion ofveins with the arteries in adults. Phosphorylation of ephrinB2
triggers angiogenesis, and downstream inhibition ofphosphorylation-dependent or PDZ domain-dependent signaling of the ephrinB2 prevents endothelial cell engraftment
and the appropriate association of endothelial cells with otherendothelial cells and with the pericytes.25
EphB4 is pathologically overexpressed in various malignant tumors, including breast cancer,33–35 lung cancer,36–38
gastric cancer,39 colorectal cancer,40,41 acute myeloid leukemia,42 cervical cancer,43 glioma,44 ovarian cancer,45,46
prostate cancer,47,48 thyroid cancer,49,50 and bladder cancer,51,52 and contributes to poor prognosis. Mutations in thephosphorylation site of the EphB4 intracellular region in lungcancer promote tumor growth in vitro.53 EphB4 gene amplification has been found and correlated with higher tumor
stage in various tumors.34,48,54 Therefore, mutations, gene
amplification, and overexpression of EphB4 contribute tomalignant progression. EphB4 is involved in cell proliferation, migration, differentiation, and angiogenesis in various
cancers.55
Several data have been reported on the forward and reverse
signaling of EphB4 and ephrinB2 in tumors. EphB4 extracellular domain can induce angiogenic responses by stimulating ephrinB2 reverse signaling in cultured endothelial cells
and promote tumor growth.56 On the other hand, ephrinB2independent effects of EphB4 have also been reported intumors.42,57 Several reports have shown that high expressionof EphB4 in tumors causes activation of ligand-independent
downstream signaling, and the effect of EphB4 on cancer
malignancy in the absence of ephrinB2 has been examined.42,58 The ephrinB2 expression in breast and lung cancer
cell lines with high expression of EphB4 is very low compared to normal cell lines.37,59 Therefore, EphB4 may often
contribute to tumor malignancy by ligand-independent effects in cancer patients.
In breast cancer in particular, EphB4 is expressed endogenously in numerous cell lines,33,34 and the EphB4-positive
rate in immunohistochemical staining is high.60 In breast
cancer cell lines, the knockdown of EphB4 by small interfering RNA results in reduced cell viability, activation of
caspase-8, and induction of apoptosis.34 Furthermore, EphB4
knockdown in a mouse breast cancer xenograft model resulted in significantly smaller tumors, induced apoptosis, andreduced tumor vascular distribution.34 Ding et al. reported
that overexpression of EphB4 caused gain-of-function activity in HER2-positive breast cancer cell lines and resistanceto lapatinib, a dual HER2/epidermal growth factor receptor
inhibitor.61 Comparison of EphB4 mRNA expression levels
in four different clinicopathological surrogate intrinsic sub-167

types of breast cancer revealed that EphB4 transcription issignificantly higher in HER2-positive breast cancer and
TNBC than luminal breast cancer.62
EphB4 could be a useful diagnostic target for prognostication and therapeutic targets of breast cancers. Therefore, aspecific monoclonal antibody (mAb) against EphB4 is needed to facilitate and ensure the diagnosis of breast cancer
subtype and to determine treatment strategies. In this study,
we report the establishment of a specific mAb against EphB4
for flow cytometric, Western blot, and immunohistochemical
analyses.
Materials and Methods
Plasmid preparation

Synthesized DNA encoding human EphB4 (Accession No.
NM_004444; Catalog No.: RC208559) was purchased from
OriGene Technologies, Inc. (Rockville, MD, USA). The
signal sequence was excluded and the EphB4 open reading
frame with an N-terminal PA16 tag or an N-terminal MAP
tag was subcloned into a pCAG-Ble vector (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan) using an InFusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan); therecombinant expression vectors were named as pCAG/PA16EphB4 and pCAG/MAP-EphB4 DNA with an N-terminal
PA16 and with an N-terminal MAP tag, respectively. The
amino acid sequences of each protein tag are as follows:
PA16 tag, 16 amino acids (GLEGGVAMPGAEDDVV), and
MAP tag,63 12 amino acids (GDGMVPPGIEDK).64 The
clone NZ-1 (rat IgG2a, lambda) was used for PA16 tag,65 andclone PMab-1 (rat IgG2a, kappa) was used for MAP tag.64
Cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) were obtained from the American Type Culture
Collection (ATCC, Manassas, VA, USA). MCF-7 was obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku
University (Miyagi, Japan). CHO-K1 cells that overexpress PA16-EphB4 (CHO/EphB4) and MAP-EphB4 (CHO/
MAP-EphB4) were generated by transfection of pCAG/
PA16-EphB4 and pCAG/MAP-EphB4 to CHO-K1 cells,
respectively, using Lipofectamine LTX Reagent (Thermo
Fisher Scientific, Inc., Waltham, MA, USA). Stable transfectants were sorted using a cell sorter (SH800; Sony Corp.,
Tokyo, Japan) and cultivated in a medium containing
0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA, USA). The
MCF-7/EphB4-knockout (KO) cell line (BINDS-52) was
generated by transfection using CRISPR/Cas9 plasmids(Assay ID: CRISPR710315_SGM) that target EphB4(Thermo Fisher Scientific, Inc.). Plasmids were transfectedusing a Neon Transfection System (Thermo Fisher Scientific,Inc.). Stable transfectants were sorted using a cell sorter(SH800).
CHO-K1, P3U1, CHO/EphB4, CHO/MAP-EphB4, MCF7, and BINDS-52 cells were cultured in a Roswell Park
Memorial Institute (RPMI)-1640 medium (Nacalai Tesque,Inc., Kyoto, Japan). All media were supplemented with 10%
heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin, 100 lg/mL of streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai Tesque,168Inc.). All cells were cultured in a humidified incubator at
37C, 5% CO2, and 95% air.

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Hybridoma production

Female BALB/c mice (5 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). The animals were housed under
specific pathogen-free conditions. All animal experiments
were conducted by relevant guidelines and regulations tominimize the pain and suffering of animals in the laboratory.
Animal experiments were approved by the Animal Care and
Use Committee of Tohoku University (Permit No.:
2022MdA-001). Mice were monitored daily for health during
the full 5-week duration of the experiment. A reduction of
more than 25% of total body weight was defined as a humane
endpoint. For splenectomy, mice were euthanized by cervicaldislocation and death was confirmed by respiratory arrest andcardiac arrest.
To develop mAbs against EphB4, we used Cell-Based
Immunization and Screening (CBIS) method.66–75 Briefly,
two BALB/c mice were injected intraperitoneally with amixture of CHO/MAP-EphB4 cells (1 · 108/mouse) with
Imject Alum (Thermo Fisher Scientific, Inc.) as adjuvant onlyat the first immunization; the second immunization was
performed 1 week after the first, and the third to fifth immunizations were performed at the same interval. Two days
after the fifth immunization as boost immunization, mice
were sacrificed and splenocytes were collected. The harvested spleen cells were subsequently fused with P3U1 cells
using Polyethylene Glycol (PEG) 1500 (Roche Diagnostics,
Indianapolis, IN, USA), and the hybridomas were grown inan RPMI-1640 medium supplemented with hypoxanthine,aminopterin, and thymidine for selection (Thermo Fisher
Scientific, Inc.) on ten 96-well plates. Culture supernatants
were screened by flow cytometry as described below.
Flow cytometry

Cultured cells were collected after brief exposure to 0.25%
trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA;
Nacalai Tesque, Inc.). They were once washed with 0.1%
bovine serum albumin (BSA) in phosphate-buffered saline(PBS) and treated with primary mAbs for 30 minutes at 4C.
Fluorescein isothiocyanate-conjugated anti-EphB4 mAb(clone: 04) was purchased from Abcam (Cambridge, MA,
USA). After washing cells twice with 0.1% BSA in PBS, thecells were then treated with Alexa Fluor 488-conjugated antimouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 minutes at 4C. After three washes,
cells were diluted with 100 lL and shaken before measurement. Fluorescence data were collected using the SA3800
Cell Analyzer (Sony Corp.). The collected data were analyzed with FlowJo software (FlowJo, OR, USA).
Determination of dissociation constantby flow cytometry

CHO/EphB4 and MCF-7 cells were suspended in 100 lL
of B4Mab-7 (0.006–50 lg/mL) serially diluted 1/2-fold, andexposed for 30 minutes, and the cells were washed twice with
0.1% BSA in PBS. Then, 50 lL of Alexa Fluor 488conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Inc.) was added and exposed for 30 minutes. Afterwashing twice, the cells were transferred to a 96-well plate

NANAMIYA ET AL.

for measurement. Fluorescence data were collected using theSA3800 Cell Analyzer. The collected data were analyzed
with FlowJo software. The dissociation constant (KD) was
calculated by fitting binding isotherms to built-in, one-sitebinding models in GraphPad PRISM 8 (GraphPad Software,Inc., La Jolla, CA, USA).
Western blot analysis

Cell lysates were extracted from cultured cells using 1%
Triton X-100, and cell remnants were removed by centrifugation. The cell lysates (10 lg) were boiled in sodium dodecyl sulfate sample buffer (Nacalai Tesque, Inc.). These
proteins were then electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation)and transferred onto polyvinylidene difluoride (PVDF)
membranes (Merck KGaA, Darmstadt, Germany).
After blocking with 4% skim milk (Nacalai Tesque, Inc.)for 1 hour, membranes were incubated with 1 lg/mL of
B4Mab-7 or 1 lg/mL of an anti-isocitrate dehydrogenase 1(IDH1) mAb, RcMab-1,76 for 30 minutes. Then, the membranes were incubated with peroxidase-conjugated anti-mouse
immunoglobulins (diluted 1:2000; Agilent Technologies, Inc.,
Santa Clara, CA, USA), or anti-rat immunoglobulins (diluted
1:10,000; Sigma-Aldrich Corp., St. Louis, MO, USA) for 30
minutes, respectively. Finally, the protein bands were visualized using ImmunoStar LD (FUJIFILM Wako Pure Chemical
Corporation) and Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan). All Western blot procedures were performed at room
temperature, and all primary and secondary antibodies werediluted with 4% skim milk.
Immunohistochemical analysis

A formalin-fixed paraffin-embedded (FFPE) breast cancer
tissue microarray (Cat. No.: T8235721-5, Lot. No.: B904111)
containing 63 cases was purchased from BioChain Institute,
Inc. (Newark, CA, USA). The antigen retrieval was performed using a direct autoclave treatment at 121C for 20
minutes in EnVision FLEX Target Retrieval Solution HighpH (Agilent Technologies, Inc.), Peroxidase blocking was
performed by treatment with 3.0% H2O2 for 10 minutes.
Blocking was then conducted using SuperBlock Blocking
Buffer (Thermo Fisher Scientific, Inc.).
Sections were then incubated with 10 lg/mL of B4Mab-7(diluted by SuperBlock Blocking Buffer) or buffer control for
1 hour at room temperature, followed by treatment with an
Envision+ kit (Agilent Technologies, Inc.) for 30 minutes.
Antigen–antibody complexes were visualized using 3,3¢diaminobenzidine tetrahydrochloride solution (Agilent
Technologies, Inc.) for 2 minutes, and sections were thencounterstained with hematoxylin (FUJIFILM Wako Pure
Chemical Corporation) for 5 minutes. The intensity ofstaining was evaluated as 0, 1+, 2+, or 3+. Hematoxylin andeosin (H&E) staining (FUJIFILM Wako Pure Chemical
Corporation) was performed using consecutive tissue sections at room temperature for 5 and 10 minutes, respectively.
Results
Establishment of anti-EphB4 mAbs

To establish anti-EphB4 mAbs, we employed the CBIS
method, using stable transfectants for both immunizations andDEVELOPMENT OF ANTI-EPHB4 MAB

flow cytometric screening (Fig. 1). Two BALB/c mice were
immunized with CHO/MAP-EphB4 cells. Hybridomas werethen seeded onto 96-well plates, and culture supernatants
positive for CHO/EphB4 and negative for CHO-K1 were selected by flow cytometry. The positive count was 54/956 wells(5.65%), which were cloned by limiting dilution. After several
additional screenings, an anti-EphB4 mAb, B4Mab-7 (IgG1,
kappa), was finally established. Subsequently, the specificity
and usefulness of B4Mab-7 were evaluated by flow cytometry,
Western blot, and immunohistochemical analyses.
Flow cytometric analyses

CHO/EphB4 cells in a dose-dependent manner (Fig. 2A),
whereas it did not react with parental CHO-K1 cells(Fig. 2B). B4Mab-7 also showed a concentration-dependent
reactivity to breast cancer cell line, MCF-7 (Fig. 2C). These
results indicated that B4Mab-7 recognizes not only exogenous EphB4 but also endogenous EphB4 on MCF-7 cells.
To confirm the specificity of B4Mab-7 against MCF-7, weestablished EphB4-knockout MCF-7 (BINDS-52) by a
CRISPR/Cas9 system, and performed flow cytometry using
B4Mab-7 against BINDS-52. B4Mab-7 showed no reaction
against BINDS-52 cells (Fig. 2D). We also confirmed that acommercially available mAb (clone 04) showed similar reactivity to CHO/EphB4 and MCF-7 cells, and never recognized BINDS-52 cells (Supplementary Fig. S1). These results
suggest that B4Mab-7 is specific for EphB4 on MCF-7 cells.

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We next examined the B4Mab-7 reactivity against breast
cancer cell lines in flow cytometry. B4Mab-7 reacted with

169

FIG. 1. Production of anti-EphB4 mAbs. The procedure of CBIS method. (A) The EphB4-overexpressed cell lines were
immunized into BALB/c mice by intraperitoneal injection. (B) Mouse spleens were harvested, fused with splenocytes andmyeloma cells, and seeded into 96-well plates. (C) Hybridoma screening was performed using flow cytometry to selectwells that did not react with the parental cell lines, but with the EphB4-overexpressed cell lines. (D) Positive wells werecloned by limiting dilution. The specificity was confirmed using an EphB4-knockout breast cancer cell line. CBIS, CellBased Immunization and Screening.

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170

NANAMIYA ET AL.

FIG. 2. Flow cytometry using B4Mab-7. (A–D) CHO/EphB4, CHO-K1, MCF-7, and BINDS-52 (EphB4-knockout MCF-7)
were treated with 0.1 or 1 mg/mL of B4Mab-7, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG. Filled,
negative control. CHO, Chinese hamster ovary.

Then we examined whether B4Mab-7 is also useful inWestern blot analysis. B4Mab-7 detected strong signals of
100–130-kDa in CHO/EphB4 and MCF-7 cells, but not in
CHO-K1 and EphB4-knockout MCF-7 (BINDS-52) cells(Fig. 4A). These results suggest that B4Mab-7 specificallyand sensitively recognizes endogenous EphB4 in breast
cancer cell lines on Western blot analysis.

sections.33–35 As depicted in Figure 5A, D, G, J, M, and P,
B4Mab-7 strongly stained the plasma membrane of breast
cancer cells, and weak staining was also observed in thecytoplasm. B4Mab-7 stained the membranes of cancer cells
in 41 of 63 (65.1%) breast cancers (Fig. 5A, D: score 3+,
Fig. 5G, J: score 2+, Fig. 5M, P: score 0; scores were divided
by the intensity of membrane staining). Treatment with
control buffer (Fig. 5B, E, H, K, N, and Q) and H&E staining(Fig. 5C, F, I, L, O, and R) were performed on consecutive
breast cancer tissues.
B4Mab-7 also stained venous or lymphatic endothelial
cells (Supplementary Fig. S2B, arrows), although it did notstain stromal tissues. These results indicate that B4Mab-7 isuseful for immunohistochemical analysis of FFPE tumor
sections. All staining results of B4Mab-7 for breast cancers
are shown in Table 1. B4Mab-7 could stain invasive ductal
carcinomas, invasive lobular carcinoma, adenocarcinoma,
fibroadenoma, and medullary carcinoma (Table 2).

Immunohistochemical analysis against breast cancers

Discussion

We next investigated whether B4Mab-7 applies to immunohistochemical analysis against FFPE breast cancer

In this study, we successfully developed a specific antiEphB4 mAb, B4Mab-7, which can be adapted to any

Determination of the binding affinity of B4Mab-7

To investigate the binding ability of B4Mab-7, the apparent KD of B4Mab-7 for CHO/EphB4 and MCF-7 wasevaluated using flow cytometry. The KD values of B4Mab-7
for CHO/EphB4 and MCF-7 were 2.9 · 10-9 M and
1.3 · 10-9 M, respectively (Fig. 3). These results indicatedthat B4Mab-7 possesses a high binding affinity for
CHO/EphB4 and MCF-7 cells.
Western blot analysis

FIG. 3. Determination of the binding affinity of B4Mab-7. (A, B) CHO/EphB4 and MCF-7 cells were suspended in
100 mL of serially diluted B4Mab-7 (6 ng/mL to 50 mg/mL). Alexa Fluor 488-conjugated anti-mouse IgG was then added.
Fluorescence data were collected using the SA3800 cell analyzer.

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DEVELOPMENT OF ANTI-EPHB4 MAB

171

FIG. 4. Western blot analysis using B4Mab-7. Cell lysates(10 mg) were electrophoresed, and proteins were transferred
onto PVDF membranes. After blocking, membranes wereincubated with 1 mg/mL of B4Mab-7 (A) or 1 mg/mL of ananti-IDH1 mAb (clone: RcMab-1) (B). The membranes
were then incubated with peroxidase-conjugated anti-mouse
immunoglobulins and rat immunoglobulins, respectively.
IDH1, isocitrate dehydrogenase 1; PVDF, polyvinylidene
difluoride.

experimental methods. The CBIS method does not need purified proteins for immunization and screening, and could
target the three-dimensional structure of transmembrane proteins. B4Mab-7 did not react with EphB4-knockout MCF-7(BINDS-52), as shown in Figure 2D, although Eph family
proteins have been reported to be expressed in MCF-7 cells inseveral studies.77,78 Fox and Kandpal also demonstrated that
other Eph family RNAs were expressed in MCF-7.79 These
results indicate that B4Mab-7 might not react with other Ephfamily proteins. In the future study, we will check whether
B4Mab-7 could crossreact with other Eph family proteins one
by one using flow cytometry.
Many anti-EphB4 mAbs are now commercially available,
as shown in Supplementary Table S1. As listed, anti-EphB4
mAbs are usually applicable for only one or two experimental
techniques. Therefore, B4Mab-7 is the first anti-EphB4 mAb,
which is useful for flow cytometry, Western blot, and IHC.
B4Mab-7 was developed using the CBIS method, whereas
almost all mAbs were developed using immunization of recombinant proteins or peptides; therefore, the CBIS method
using EphB4-overexpressed cell lines, as in this study, might
be the best way to obtain useful anti-EphB4 mAbs. The CBIS
method could be advantageous for the development of mAbs
to the other Eph receptors.
Although many mAbs against anti-Eph receptors, such as
anti-EphA2 mAbs,80–94 anti-EphA3 mAbs,95–102 antiEphA10 mAbs,103 anti-EphB2 mAbs,104 and anti-EphB4
mAbs,105,106 have been developed, almost all mAbs have notbeen used clinically,107 excluding only a few anti-Eph receptor mAbs.92,93,98 1C1 was a promising anti-EphA2 mAb
that showed agonist activity and caused phosphorylation and

FIG. 5. Immunohistochemical analysis by B4Mab-7 forbreast cancers. After antigen retrieval using EnVision FLEX
Target Retrieval Solution High pH, tissue arrays were incubated with 10 mg/mL of primary B4Mab-7 for 1 hour at room
temperature, followed by treatment with Envision+ kit for 30
minutes. The color was developed using 3,3-diaminobenzidine
tetrahydrochloride for 2 minutes, and sections were thencounterstained with hematoxylin. (A, D) The 3+ staining
pattern (case 1), (B, E) The buffer control (case 1), (C, F) The
H&E staining (case 1). (G, J) The 2+ staining pattern (case 2),(H, K) The buffer control (case 2), (I, L) The H&E staining(case 2), (M, P) The negative staining pattern (case 3), (N, Q)
The buffer control (case 3), (O, R) The H&E staining (case 3).
Scale bar = 100 mm. H&E, hematoxylin and eosin.
internalization of EphA2.90 Since 1C1 itself showed no antitumor effect, MEDI-547, an antibody-drug conjugate(ADC) composed of the cytotoxic drug auristatin (toxin)
linked to 1C1, was created.90 MEDI-547 inhibited tumor
growth and metastasis in vivo. Although MEDI-547 was
moved to phase I clinical study, it was terminated due toserious side effects such as treatment-related bleeding, coagulation events, and elevated liver enzymes at the starting
dose in 3/6 patients.93
IIIA4/KB004/ifabotuzumab is an anti-EphA3 mAb currently in development for the treatment of glioblastoma
multiforme.102 The phase I study of IIIA4 has been completed and the safety has been confirmed.108 Interestingly, theimmunogen for IIIA4 uses LK63 human pre-B acuteDownloaded by Yukinari Kato from www.liebertpub.com at 10/30/23. For personal use only.

Table 1. Results of B4Mab-7 Immunostaining in 63 Patients with Breast Cancers
No.

Sex

Age

Organ

Pathology diagnosis

Differentiation

TNM

B4Mab-7

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63

F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F

44
58
40
52
60
57
48
66
58
63
32
59
44
60
44
82
58
57
41
44
78
60
/
46
41
59
45
43
26
40
38
51
45
45
47
55
58
47
38
40
57
42
60
58
41
50
60
53
65
43
57
37
50
48
50
53
49
65
43
58
48
/
/

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Invasive ductal carcinoma
Invasive ductal carcinoma
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Invasive ductal carcinoma
Invasive ductal carcinoma
Adenocarcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive lobular carcinoma
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Invasive ductal carcinoma
Adenocarcinoma
Invasive ductal carcinoma
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Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Fibroadenoma
Invasive ductal carcinoma
Fibroadenoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma
Invasive ductal carcinoma

Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Well
Well
Moderately
Moderately
Moderately
Moderately
PoorlyModerately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
PoorlyPoorly
N/A
N/A
N/A
N/A
Moderately
PoorlyPoorlyModerately
Moderately
Moderately
Moderately
PoorlyPoorlyPoorlyModerately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
PoorlyModerately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately
Moderately

T2N2M1
T2N2M1
T2N1M0T2N2M1
T2N1M1
T2N0M0T2N0M0T2N0M0T2N2M1
T2N0M0T2N0M0T2N2M0T2N2M0T2N1M0T2N2M0T2N1M1
T2N1M1
T3N3M0T2N1M0T2N2M0T2N1M0T2N0M0T2N1M1
T2N3M1
T2N2M0T2N0M0T2N0M0T2N1M1
T1N0M0T1N0M0T2N0M0T2N2M0T2N0M0T2N1M0T2N1M0T2N3M1
T3N3M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N2M1
T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N3M0T2N1M0T2N0M0T2N0M0T2N0M0T2N1M0T2N0M0T2N0M0T2N0M0TxNxMxTxNxMx

2+
3+
1+
0
1+
3+
2+
0
0
1+
1+
0
0
1+
3+
1+
2+
1+
3+
0
1+
1+
1+
0
0
1+
0
0
2+
0
1+
0
1+
2+
0
2+
1+
3+
1+
2+
0
2+
2+
0
0
1+
0
3+
0
2+
2+
1+
0
0
1+
3+
2+
0
2+
0
1+
2+
1+

172

DEVELOPMENT OF ANTI-EPHB4 MAB

173

Table 2. Result of B4Mab-7 Immunostaining in 63 Patients with Breast Cancers
B4Mab-7 immunostaining
Pathology diagnosis

Downloaded by Yukinari Kato from www.liebertpub.com at 10/30/23. For personal use only.

Invasive ductal carcinoma
Invasive lobular carcinoma
Adenocarcinoma
Fibroadenoma
Medullary carcinoma

No. of cases

3+

2+

1+

0

No. of positive cases

55
3
2
2
1

6
0
0
0
1

12
0
1
1
0

18
1
0
1
0

19
2
1
0
0

36/55 (65.4%)
1/3 (33.3%)
1/2 (50.0%)
2/2 (100%)
1/1 (100%)

lymphoblastic leukemia cells,109 and it is the only antiEphA3 mAb that is adaptable to flow cytometry, Western
blot, and IHC like B4Mab-7.109 This fact supports the suitability of immunization using cell lines for the developmentof useful anti-Eph receptor mAbs.
Because Eph receptors are expressed in normal tissues andhave important physiological functions, targeting them withconventional antibody drugs has been difficult. Therefore,
recently, naked antibodies have been applied to chimericantigen receptor-T cell therapy,110,111 ADC,90,91,102,112 bispecific T cell engager,103,113 bispecific antibody,114,115 andliposomes,116 to target Eph receptors. Bispecific antibodies
against EphA2/EphA3 reduced the clonogenicity of recurrent
glioblastoma in vitro and the tumorigenic potential of xenograft recurrent glioblastoma in vivo.115
In conclusion, we established an anti-EphB4 mAb,
B4Mab-7, which is applicable for flow cytometry, Western
blot, and IHC. In future studies, the heavy chain subclass of
B4Mab-7 should be modified from IgG1 to IgG2a to enhance
effector function (antibody-dependent cellular cytotoxicity
and complement-dependent cytotoxicity) and its antitumor
effect on breast cancer in vitro and in vivo should be investigated.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported by Grant-in-Aid for the Japan
Society for the Promotion of Science ( JSPS) Fellows Grant
No. 22J20977 (to R.N.), JST SPRING, and Grant No.
JPMJSP2114 (to R.N.). This research was also supported, inpart, by the Japan Agency for Medical Research and Development (AMED) under Grant Nos. JP22ama121008 (to Y.K.),
JP22am0401013 (to Y.K.), 23bm1123027h0001 (to Y.K.),
JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.).
Supplementary Material

Supplementary Figure S1
Supplementary Figure S2
Supplementary Table S1References

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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: September 1, 2023
Accepted: September 18, 2023