A Sensitive Monoclonal Antibody B4Mab-7 against EphB4 Was Developed for Breast Cancers

1. 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 all cancer cases) and estimated 684,996 deaths (6.9% of all cancer 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 [2,3,4]. Breast cancer treatment currently includes surgery, radiation therapy, chemotherapy, and molecular targeted therapy [5,6,7].

Molecular targeted therapy depends on the subtype of breast 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 [8,9,10]. Breast cancer subtypes can be largely classified into hormone receptor (ER or PR)-positive luminal A, luminal B, HER2-positive, and triple-negative breast cancer (TNBC) [8,9,10].

The erythropoietin-producing hepatocellular carcinoma (Eph) receptors belong to the largest family of receptor tyrosine kinases in mammals [11]. Eph receptors are physiologically associated 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 a ligand binding domain, followed by a cysteine-rich domain (including the sushi and epidermal growth factor-like domains) and two fibronectin domains [15,16]. The intracellular side of the Eph receptor consists of a transmembrane region, a tyrosine 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 cross-talk between the groups [17,18,19,20,21,22,23]. In contrast to other Eph receptors, EphB4 shows clear specificity for the ligand ephrinB2 and very weak binding affinity for ephrinB1 and ephrinB3 [16].

EphB4 and ephrinB2 are biologically essential for cell adhesion, cell migration, stabilization, branching, and remodeling during embryogenesis for cardiovascular formation [24,25,26,27,28,29,30]. Mice lacking either EphB4 (expressed in veins) or ephrinB2 (expressed in arteries) showed identical defects in the formation of capillary connections between the network of arteries and veins in the head and yolk sac, resulting in embryonic lethal [29,30,31,32]. EphB4 and ephrinB2 create a repulsive effect between them and act to prevent the fusion of the veins with the arteries in adults. Phosphorylation of ephrinB2 triggers angiogenesis, and downstream inhibition of phosphorylation-dependent or PDZ domain-dependent signaling of the ephrinB2 prevents endothelial cell engraftment and prevents the appropriate association of endothelial cells with other endothelial cells and with the pericytes [25].

EphB4 is pathologically overexpressed in various malignant tumors, including breast cancer [33,34,35], lung cancer [36,37,38], 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], bladder cancer [51,52], and contributes to poor prognosis. Mutations in the phosphorylation site of the EphB4 intracellular region in lung cancer 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 to malignant 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, ephrinB2-independent effects of EphB4 have also been reported in tumors [42,57]. Several reports have shown that high expression of 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]. 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, and reduced 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 resistance to lapatinib, a dual HER2/epidermal growth factor receptor inhibitor [61]. Comparison of EphB4 mRNA expression levels in four different clinicopathological surrogate intrinsic subtypes of breast cancer revealed that EphB4 transcription is significantly higher in HER2-positive breast cancer and TNBC than luminal breast cancer [62].

Highly sensitive and specific monoclonal antibodies (mAbs) are needed to facilitate and ensure the diagnosis of breast cancer subtype and to determine treatment strategies. EphB4 may be a useful diagnostic target for prognostication and therapeutic targets of breast cancers, requiring the development of sensitive and specific mAb. In this study, we report the establishment of sensitive and specific mAbs against EphB4 for flow cytometric, western blot, and immunohistochemical analyses.

2. Materials and methods

2.1. 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 In-Fusion HD Cloning Kit (Takara Bio, Inc. Shiga, Japan); the recombinant expression vectors were named as pCAG/PA16-EphB4 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 IgG_2a, lambda) was used for PA16 tag [65], and clone PMab-1 (rat IgG_2a, kappa) was used for MAP tag [64].

2.2. Cell lines

Chinese hamster ovary (CHO)-K1, P3X63Ag8U.1 (P3U1), and MDA-MB-468 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) by a Neon Transfection System (Thermo Fisher Scientific, Inc.). 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 transfected, using Lipofectamine LTX Reagent (Thermo Fisher Scientific, Inc.) by 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, MCF-7, and BINDS-52 cells were cultured in a Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan). MDA-MB-468 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Nacalai Tesque, Inc.). All media were supplemented with 10% heat-inactivated 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.). All cells were cultured in a humidified incubator at 37°C, 5% CO_2, and 95% air.

2.3. Hybridoma production

Female BALB/c mice (five 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 to minimize the pain and suffering of animals in the laboratory. Animal experiments were approved by the Animal Care and Use Committee of Tohoku University (Permit number: 2022MdA-001). Mice were monitored daily for health during the full five-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 cervical dislocation and death was confirmed by respiratory arrest and cardiac arrest.

To develop mAbs against EphB4, we used CBIS method [66,67,68,69,70,71,72,73,74,75]. Briefly, two BALB/c mice were injected intraperitoneally with a mixture of CHO/MAP-EphB4 cells (1 × 10⁸/mouse) with Imject Alum (Thermo Fisher Scientific, Inc.) as adjuvant only at the first immunization; the second immunization was performed one 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 in an 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.

2.4. 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 min at 4°C. 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, the cells were then treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 4°C. After three washes, cells were diluted with 100 μL 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). 0.1% BSA in PBS was used to dilute the antibodies.

2.5. Determination of dissociation constant (K_D) by flow cytometry

CHO/EphB4, MCF-7, and MDA-MB-468 cells were suspended in 100 μL of B4Mab-7 (0.006-50 μg/mL) serially diluted 1/2-fold, and exposed for 30 min, and the cells were washed twice with 0.1% BSA in PBS. Then, 50 μL of Alexa Fluor 488-conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Inc.) was added and exposed for 30 minutes. After washing twice, the cells were transferred to a 96-well plate for measurement. Fluorescence data were collected using the SA3800 Cell Analyzer. The collected data were analyzed with FlowJo software. The dissociation constant (K_D) was calculated by fitting binding isotherms to built-in, one-site binding models in GraphPad PRISM 8 (GraphPad Software, Inc., La Jolla, CA, USA). 0.1% BSA in PBS was used to dilute the antibodies.

2.6. 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 μg) 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 h, membranes were incubated with 1  μg/mL of B4Mab-7 or 1  μg/mL of an anti-isocitrate dehydrogenase 1 (IDH1) mAb, RcMab-1 [76], for 30 min. 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 min, 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 were diluted with 4% skim milk.

2.7. 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 121°C for 20 min in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) Peroxidase blocking was performed by treatment with 3.0% H₂O₂ for 10 min. Blocking was then conducted using SuperBlock Blocking Buffer (Thermo Fisher Scientific, Inc.). Sections were then incubated with 10  μg/mL of B4Mab-7 (diluted by SuperBlock Blocking Buffer) or buffer control (SuperBlock Blocking Buffer) for 1 h at room temperature, followed by treatment with an Envision+ kit (Agilent Technologies, Inc.) for 30 min. Antigen–antibody complexes were visualized using 3,3′-diaminobenzidine tetrahydrochloride solution (Agilent Technologies, Inc.) for 2 min, and sections were then counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation) for 5 min. The intensity of staining was evaluated as 0, 1+, 2+, or 3+. Hematoxylin and eosin (H&E) staining (FUJIFILM Wako Pure Chemical Corporation) was performed using consecutive tissue sections at room temperature for 5 and 10 min, respectively.

3. Results

3.1. Establishment of anti-EphB4 mAbs

To establish anti-EphB4 mAbs, we employed the Cell-Based Immunization and Screening (CBIS) method, using stable transfectants for both immunizations and flow cytometric screening (Figure 1). Two BALB/c mice were immunized with CHO/MAP-EphB4 cells. Hybridomas were then 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 blotting, and immunohistochemical analyses.

3.2. Flow cytometric analyses

We next examined the B4Mab-7 reactivity against breast cancer cell lines in flow cytometry. B4Mab-7 reacted with CHO/EphB4 cells in a dose-dependent manner (Figure 2A), whereas it did not react with parental CHO-K1 cells (Figure 2B). B4Mab-7 also showed a concentration-dependent reactivity to breast cancer cell lines, MCF-7 and MDA-MB-468 (Figure 2C,D). These results indicated that B4Mab-7 recognizes not only exogenous EphB4 but also endogenous EphB4 in breast cancer cell lines.

To confirm the specificity of B4Mab-7 against MCF-7, we established 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 (Figure 2E). We also confirmed that EphB4 was completely knocked out in BINDS-52 cells by commercially available mAb (clone 04). (Supplementary Figure 1) These results suggest that B4Mab-7 is specific for EphB4 of MCF-7.

3.3. Determination of the binding affinity of B4Mab-7

To investigate the binding ability of B4Mab-7, the apparent dissociation constant (K_D) of B4Mab-7 for CHO/EphB4, MCF-7, and MDA-M468 was evaluated using flow cytometry. The K_D values of B4Mab-7 for CHO/EphB4, MCF-7, and MDA-MB-468 were 2.9 × 10^-9 M, 1.3 × 10^-9 M, and 3.3 × 10^-9 M (Figure 3), respectively, indicating that B4Mab-7 possesses a high binding affinity for CHO/EphB4, MCF-7, and MDA-M468 cells.

3.4. Western blot analysis

Then, we examined whether B4Mab-7 is also useful in western blot analysis. B4Mab-7 detected strong signals of 100~130-kDa in CHO/EphB4, MCF-7, and MDA-MB-468 cells, but not in CHO-K1 cells (Figure 4A). These results suggest that B4Mab-7 specifically and sensitively recognizes endogenous EphB4 in breast cancer cell lines on western blot analysis.

3.5. Immunohistochemical analysis against breast cancers

We next investigated whether B4Mab-7 applies to immunohistochemical analysis against formalin-fixed paraffin-embedded (FFPE) breast cancer sections [33,34,35]. As depicted in Figure 5A, D, G, J, M, P, B4Mab-7 strongly stained the plasma membrane of breast cancer cells, and weak staining was also observed in the cytoplasm. B4Mab-7 stained the membranes of cancer cells in 41 of 63 (65.1%) breast cancers (Figure 5A,D: score 3+, Figure 5G,J: score 2+, Figure 5M, P: score 0; Scores were divided by the intensity of membrane staining). Treatment with control buffer (Figure 5B, E, H, K, N, Q) and hematoxylin and eosin (H&E) staining (Figure 5C,F,I,L,O,R) were performed on consecutive breast cancer tissues.

B4Mab-7 also stained venous or lymphatic endothelial cells (Supplementary Figure 2), although it did not stain stromal tissues. These results indicate that B4Mab-7 is useful 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).

4. Discussion

In this study, we successfully developed a highly sensitive and specific anti-EphB4 mAb, B4Mab-7, which can be adapted to any experimental method using the CBIS method (Figure 1). The CBIS method does not need purified proteins for immunization and screening, and could target the three-dimensional structure of transmembrane proteins.

Many anti-EphB4 mAbs are now commercially available as shown in Supplementary Table 1. 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 blotting, and immunohistochemistry. 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 [77,78,79,80,81,82,83,84,85,86,87,88,89,90,91], anti-EphA3 mAbs [92,93,94,95,96,97,98,99], anti-EphA10 mAbs [100], anti-EphB2 mAbs [101], anti-EphB4 mAbs [102,103] have been developed, almost all mAbs have not been used clinically [104], excluding only a few anti-Eph receptor mAbs [89,90,95]. 1C1 was a promising anti-EphA2 mAb that showed agonist activity and caused phosphorylation and internalization of EphA2 [87]. 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 [87]. MEDI-547 inhibited tumor growth and metastasis in vivo. Although MEDI-547 was moved to phase I clinical study, it was terminated due to serious side effects such as treatment-related bleeding, coagulation events, and elevated liver enzymes at the starting dose in 3/6 patients [90]. IIIA4/KB004/Ifabotuzumab is an anti-EphA3 mAb currently in development for the treatment of glioblastoma multiforme [99]. The phase I study of IIIA4/KB004/Ifabotuzumab has been completed and the safety has been confirmed [105]. Interestingly, the immunogen for IIIA4 uses LK63 human pre-B acute lymphoblastic leukemia cells [106], and it is the only anti-EphA3 mAb that is adaptable to flow cytometry, western blotting, and immunohistochemistry like B4Mab-7 [106]. This fact supports the suitability of immunization using cell lines for the development of useful anti-Eph receptor mAbs.

Because Eph receptors are expressed in normal tissues and have important physiological functions, targeting them with conventional antibody drugs has been difficult. Therefore, recently, naked antibodies have been applied to chimeric antigen receptor-T cell therapy [107,108], ADC [87,88,99,109], bispecific T-cell engager [100,110], bispecific antibody [111,112], and liposomes [113], 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 [112].

In conclusion, we established an anti-EphB4 mAb, B4Mab-7, which is applicable for flow cytometry, western blotting, and immunohistochemistry. In the future study, the heavy chain subclass of B4Mab-7 should be modified from IgG₁ to IgG_2a 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.