An Anti-Human EphA2 Monoclonal Antibody Ea₂Mab-7 Shows High Sensitivity for Flow Cytometry, Western Blot, and Immunohistochemical Analyses

1. Introduction

Ephrin receptors (Eph) have a single transmembrane domain and are grouped into A and B categories based on their extracellular domains, which determine the binding affinity for ligands [1,2]. The interaction of Eph with the membrane-bound ephrin-A family ligands mediates communication between cells of the same or different types, and contact-dependent bidirectional (forward and reverse) signaling spreads from adjacent cells to neighboring cells [3,4,5]. The bidirectional signaling of the Eph system plays critical roles in tissue development, homeostasis, and regeneration.

EphA2 has been studied extensively in tumor development and progression [6]. Abundant expression of EphA2 has been reported in various cancers such as prostate [7], lung [8], esophageal [9], colorectal [10], cervical [11], ovarian [12], skin [13], and breast cancers [14]. It is correlated with the malignancy [15]. EphA2 expression is associated with poor prognosis, elevated metastatic potential, and reduced patient survival [16,17]. Notably, the expression of EphA2 protein and mRNA was correlated with lymph node metastasis, clinical stage, and histologic grade of breast cancer [18]. Therefore, EphA2 is a promising target in cancer therapy because of its relatively low levels in most normal adult tissues [3].

Monoclonal antibody (mAb) therapy targeting EphA2 has been developed based on evidence that ligand stimulation, such as ephrin A1, is sufficient to induce EphA2 degradation through internalization [19]. For instance, anti-EphA2 mAbs (EA2 and B233) promoted EphA2 phosphorylation and degradation in cancer cells, and administration of EA2 mAb significantly decreased tumor growth in xenograft model [20]. In addition, a single-chain Fv of anti-EphA2 mAb (D2) blocked the ligand interaction in COS-7 cells and induced apoptosis in the lymphoma cell line [21]. An anti-EphA2 mAb (SHM16) does not inhibit the interaction between ephrin A1 and EphA2 [22]. However, SHM16 stimulates the internalization of EphA2 and inhibits malignant features of melanoma A375 cells. A humanized anti-EphA2 mAb (DS-8895a) was developed as a therapeutic mAb with antibody-dependent cellular cytotoxicity (ADCC). Treatment with DS-8895a inhibited tumor growth of EphA2-positive human breast and gastric cancers in xenograft model [23]. Anti-EphA2 mAb-based immunotherapy using chimeric antigen receptor-T cells has also been developed and is undergoing clinical trials [24,25].

We have developed various mAbs against membrane proteins using the Cell-Based Immunization and Screening (CBIS) method [26,27,28,29,30,31,32,33]. The mAbs obtained by this method are prone to recognize conformational epitopes and are suitable for flow cytometry. Furthermore, some of these mAbs are also available for western blot, immunocytochemistry (ICC), and immunohistochemistry (IHC). This allows simultaneous contributions to the development of both therapy and diagnosis. Among the anti-EphA2 mAbs developed to date, SHM16 is known to recognize intact structures on the cell surface, while mAbs such as D4A2 are suitable only for western blot and IHC [34,35]. Anti-EphA2 mAbs available for flow cytometry, western blot, ICC, and IHC have not been reported (Supplementary Table S1). This study employed the CBIS method to generate a highly versatile anti-EphA2 mAb.

2. Materials and Methods

2.1. Cell Lines

Chinese hamster ovary (CHO)-K1, P3X63Ag8.U1 (P3U1), human glioblastoma LN229, and human breast cancer MDA-MB-231 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The human pancreas cancer MIA PaCa-2 and colorectal cancer HCT-15 were obtained from the Cell Resource Center at Tohoku University (Miyagi, Japan). The human lung cancer PC-10 was obtained from Immuno-Biological Laboratories Co., Ltd. (Gunma, Japan).

MDA-MB-231, MIA PaCa-2, HCT-15, LN229, and LN229/EphA2 were maintained in DMEM. CHO-K1, CHO/EphA2, P3U1, and PC-10 were maintained in RPMI-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan). The media were supplied with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B (Nacalai Tesque).

2.2. Plasmid Construction and Establishment of Stable Transfectants

The genes encoding human EPHA2 (NM_004431) was subcloned into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The EphA2 overexpressed CHO-K1 and LN229 were generated using anti-EphA2 mAb, SHM16 (mouse IgG_2b, κ, BioLegend, San Diego, CA, USA) and a SH800 cell sorter (Sony Corporation, Tokyo, Japan).

2.3. Development of Hybridomas

The female BALB/cAJcl mice were purchased from CLEA Japan (Tokyo, Japan). The mice were intraperitoneally immunized with LN229/EphA2 cells (1×10⁸ cells/injection) and Alhydrogel adjuvant 2% (InvivoGen). After four additional immunizations with LN229/EphA2 cells, the hybridomas were generated by fusing the spleen cells with P3U1 cells using polyethylene glycol 1500 (Roche Diagnostics, Indianapolis, IN, USA).

2.4. Flow Cytometric Analysis

Cells were detached and harvested using 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.) to prevent enzymatic degradation of cell surface proteins. The cells were washed with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS, blocking buffer) and stained with mAbs by incubating for 30 minutes at 4°C. Subsequently, the cells were stained with Alexa Fluor 488-conjugated anti-mouse IgG (2,000-fold dilution; Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 minutes at 4°C. The data were collected using the SA3800 Cell Analyzer and analyzed using FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA).

2.5. Determination of Dissociation Constant (K_D) by Flow Cytometry

CHO/EphA2 and MDA-MB-231 cells were treated with serially diluted Ea₂Mab-7 and SHM16 (0.005–10 μg/mL). Subsequently, the cells were stained with Alexa Fluor 488-conjugated anti-mouse IgG (200-fold dilution) for 30 minutes at 4°C. The data were collected using the SA3800 Cell Analyzer and analyzed using FlowJo software. The fitting binding isotherms determined the dissociation constant (K_D) values to built-in one-side binding models of GraphPad Prism 6 (GraphPad Software, Inc. La Jolla, CA, USA).

2.6. Western Blot Analysis

Whole cell lysate (10 μg of protein per lane) was separated into 5–20% polyacrylamide gels (FJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.) in PBST, the membranes were incubated with 1 μg/mL of Ea₂Mab-7, SHM16, or an anti-β-actin mAb (clone AC-15; Sigma-Aldrich Corporation, St. Louis, MO, USA). Subsequently, they were incubated with anti-mouse IgG conjugated with horseradish peroxidase (1,000-fold dilution; Agilent Technologies, Inc., Santa Clara, CA, USA). Chemiluminescence signals were developed with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) or Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Inc.) and detected using a Sayaca-Imager (DRC Co., Ltd., Tokyo, Japan).

2.7. Immunocytochemical Analysis (ICC)

Cell blocks were prepared using iPGell (Genostaff Co., Ltd., Tokyo, Japan). The paraffin-embedded cell sections were autoclaved in a citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan). After blocking using the SuperBlock T20 Blocking Buffer (Thermo Fisher Scientific Inc.), the sections were incubated with 10 μg/mL of Ea₂Mab-7, and then treated with the Envision+ Kit (Agilent Technologies Inc.). Color was developed using 3,3’-diaminobenzidine tetrahydrochloride (Agilent Technologies Inc.), and counterstaining was performed using hematoxylin (Merck KGaA, Darmstadt, Germany).

2.8. Immunohistochemical Analysis (IHC)

Formalin-fixed paraffin-embedded (FFPE) breast invasive ductal carcinoma tissue array (Cat. No.: BR729) containing 71 cases was purchased (FUJIFILM Wako Pure Chemical Corporation). The section was stained with Ea₂Mab-7 (10 μg/mL) using BenchMark ULTRA PLUS (Roche Diagnostics, Indianapolis, IN).

3. Results

3.1. Development of Anti-Human EphA2 mAbs

Two BALB/cAJcl mice were immunized with LN229/EphA2 cells, and the generated hybridomas were seeded into 96-well plates. After forming colonies, supernatants were collected and analyzed by flow cytometry-based high throughput screening to determine supernatants that were negative for CHO-K1 and positive for CHO/EphA2. Subsequently, anti-EphA2 mAb-producing hybridomas were cloned by limiting dilution. Finally, a clone Ea₂Mab-7 (IgG₁, κ) was established (Figure 1).

3.2. Flow Cytometry

We conducted flow cytometry using Ea₂Mab-7 against CHO/EphA2 and CHO-K1 cells. Ea₂Mab-7 recognized CHO/EphA2 cells in a dose-dependent manner, ranging from 10 to 0.005 μg/mL, but did not bind to CHO-K1 cells at any concentrations (Figure 2A), indicating Ea₂Mab-7 specifically recognizes EphA2 on the cell surface. A commercially available anti-EphA2 mAb (SHM16) exhibited a similar reactivity against CHO/EphA2 and CHO-K1 cells compared to Ea₂Mab-7 (Figure 2B). We next investigated the reactivity of Ea₂Mab-7 against endogenously EphA2-expressing cell lines, MDA-MB-231, MIA PaCa-2, HCT-15, PC-10, and LN229. Ea₂Mab-7 and SHM16 reacted with these cell lines at 10 μg/mL (Figure 3).

3.3. Determination of K_D Values

We performed flow cytometry to determine the binding affinity and calculated the K_D values of Ea₂Mab-7 and SHM16. The K_D values of Ea₂Mab-7 for CHO/EphA2 and MDA-MB-231 cells were 7.7 × 10⁻⁹ M and 2.1 × 10⁻⁹ M, respectively (Figure 4). The K_D values of SHM16 for CHO/EphA2 and MDA-MB-231 were 8.4 × 10⁻⁹ M and 2.3 × 10⁻⁹ M, respectively. These results indicated that the affinity of Ea₂Mab-7 is slightly higher than that of SHM16.

3.4. Western Blot Analysis

We next investigated whether Ea₂Mab-7 is available for western blot. Whole cell lysate of CHO-K1, CHO/EphA2, MDA-MB-231, MIA PaCa-2, HCT-15, PC-10, and LN229 were also analyzed. Ea₂Mab-7 showed a clear signal around the estimated molecular weight (108 kDa) of EphA2 in CHO/EphA2 (Supplementary Figure S1A and Figure 5A). In contrast, SHM16 was not available for western blot (Supplementary Figure S1B). Furthermore, Ea₂Mab-7 is also capable of detecting endogenous EphA2 of MDA-MB-231, MIA PaCa-2, and HCT-15 (Figure 5A). Weak signal was detected in PC-10 and LN229 (Figure 5A).

3.5. ICC

To investigate whether Ea₂Mab-7 can be used for ICC analyses, paraffin-embedded CHO-K1 and CHO/EphA2 sections were stained with Ea₂Mab-7. Apparent membranous staining by Ea2Mab-7 was observed in CHO/EphA2 (Figure 6A) but not in CHO-K1 (Figure 6B). These results indicate the usefulness of Ea₂Mab-7 for detecting EphA2-positive cells in paraffin-embedded cell samples.

3.6. IHC

Next, the FFPE breast cancer tissue array was stained with Ea2Mab-7. The cytoplasmic pattern of EphA2 staining was observed. We classified the results into strong positive (2+, Figure 7A), positive (1+, Figure 7B), and negative (0, Figure 7B). The staining results were summarized in Supplementary Table S2.

4. Discussion

In this study, we established a novel anti-EphA2 mAb, Ea₂Mab-7, which is the first mAb available to various applications, including flow cytometry (Figs. 2–4), western blot (Figure 5), ICC (Figure 6), and IHC (Figure 7). We established twelve clones during the establishment of anti-EphA2 mAbs by the CBIS method. However, Ea₂Mab-7 is the sole mAb available to all applications. Although SHM16 was established by the immunization of A375 melanoma cells into mice [22], SHM16 was not available to western blot (Supplementary Figure 1). Therefore, the identification of the epitope of Ea₂Mab-7 is essential to reveal the structural basis of antigen recognition by Ea₂Mab-7. We have developed the PA insertion for epitope mapping (PAMAP) and RIEDL insertion for epitope mapping (REMAP) to determine the epitope of mAbs [36,37,38,39,40]. The epitopes of anti-mouse CD39 mAb (C₃₉Mab-1) could be determined using both REMAP and PAMAP methods [36]. Furthermore, the epitopes of anti-CD44 mAbs (C₄₄Mab-5 and C₄₄Mab-46) [37,38] and anti-EGFR mAbs (EMab-51 and EMab-134) [39,40] could be determined using the REMAP method. Therefore, further studies are required to determine the epitope of Ea₂Mab-7. If Ea₂Mab-7 possesses the linear and non-glycosylated epitope, it may contribute to developing highly versatile mAbs to other Eph families using peptide immunization of the corresponding sequence.

Interaction between EphA2 and ephrin-As anchored on the plasma membranes of adjacent cells induces the large oligomeric complexes that mediate bidirectional signaling [5]. In contrast, the ephrin-A-independent non-canonical signaling of EphA2 plays a critical role in cancer progression [3]. The serine-threonine kinases of the RSK family [41] and AKT [42] can induce the phosphorylation of EphA2 (S897), which mediates oncogenic transformation [43], metabolic reprogramming [44], invasion [45], and resistance to therapies [8,46]. Furthermore, phosphorylated EphA2 (S897) has been shown to correlate with poor prognosis of breast cancer patients [47]. Therefore, anti-EphA2 mAbs can target cancer cells, which activate the non-canonical EphA2 signaling. To target the EphA2-positive cancer cells by Ea₂Mab-7 (IgG₁), we should generate a class-switched mouse IgG_2a mAb from mouse IgG₁. Furthermore, we previously produced defucosylated IgG_2a type mAbs to enhance the ADCC and in vivo antitumor effect in mouse xenograft models [48,49]. The class-switched and defucosylated type Ea₂Mab-7 could contribute to treating EphA2-positive cancers in preclinical studies.

Focusing on the detailed signal intensities, different correlations are observed between applications. For instance, the western blot signal did not wholly match the flow cytometry signal intensity (Figure 3A and Figure 5A). Clear signals for CHO/EphA2, MDA-MB-231, MIA PaCa-2, and HCT-15 cells were detected in both flow cytometry and western blot. However, weak or nearly undetectable signals were observed in PC-10 and LN229 cells in the western blot, while clear signals were detected in flow cytometry. Western blot shows the total amount of EphA2 expression in whole cells. At the same time, flow cytometry indicates the amount of EphA2 on the cell surface, suggesting that intracellular and extracellular levels of EphA2 differ depending on the cell line. This discrepancy may help explain the malignancy of cancer cells, as the abundant expression of EphA2 on the cell surface enables the triggering of non-canonical signaling that promotes cancer cell growth [3,19,50]. Additionally, compared to CHO/EphA2 cells, the signals of the endogenously expressed cell lines show a slightly smaller molecular weight in western blot, which may be due to the alternative splicing of EphA2 [51]. Therefore, mAbs, available for various applications, such as western blot and flow cytometry, provide significant advantages in estimating intracellular and extracellular protein levels and detecting protein processing.

Ea₂Mab-7 can detect exogenous and endogenous EphA2 in ICC and IHC analysis (Figs. 6 and 7). We could detect apparent membranous staining in the CHO/EphA2 section (Figs. 6). In contrast, IHC analysis of the breast cancer tissue array mainly showed cytoplasmic staining in most cases (Figs. 7 and Supplementary Table S2). This result was consistent with the previous reports using several anti-EphA2 Abs [35,52,53]. EphA2 was shown to receive the ligand-induced internalization [54]. Furthermore, the activation of receptor tyrosine kinase MET can induce the phosphorylation of EphA2 (S897) and endosomal internalization [55]. It would be interesting to investigate whether the cytoplasmic localization of EphA2 is involved in the malignant properties of cancer cells.

In conclusion, Ea₂Mab-7 is a highly sensitive and versatile mAb for basic research and is expected to contribute to clinical applications such as antibody therapy and tumor diagnosis.