Epitope Mapping of an Anti-CD44v4 Monoclonal Antibody (C₄₄Mab-108) using Enzyme-Linked Immunosorbent Assay

1. Introduction

CD44 has various isoforms, which are generated by the alternative splicing of CD44 pre-mRNA [1]. The mRNA of CD44 standard (CD44s) isoform is produced by constant region exons, including the first five (1 to 5) and the last five (16 to 20) [2]. The mRNAs of CD44 variant (CD44v) isoform are produced by the assembling of variant exons (v1–v10) with the constant region exons of CD44s [3]. CD44s and CD44v receive the post-translational modifications, including N- or O-glycosylation [4]. Both CD44s and CD44v can attach to hyaluronic acid, which is important for cellular adhesion, homing, and motility [5].

CD44v plays important roles in the tumor progression by specific functions of variant exon-encoded regions [6]. The heparin-binding growth factors are recruited to heparan sulphate modified in the v3-encoded region [7,8]. MET, a receptor tyrosine kinase for hepatocyte growth factor, associates with the v6-encoded region [9,10]. These functions are essential for the activation of growth factor signaling and tumor proliferation. However, the roles of CD44 variant 4 (CD44v4)-encoded region have not been investigated. Therefore, specific antibodies against CD44v4 are indispensable for basic research, tumor diagnosis, and therapy.

We have established anti-CD44 monoclonal antibodies (mAbs), which recognize the standard [11,12]- or each variant [13,14,15,16,17,18,19,20]-encoded region. All mAbs can be used for flow cytometry, western blotting, and immunohistochemistry, and are expected to contribute not only to basic research but also to pathogenic diagnosis. We also determined the critical epitopes of C₄₄Mab-5 [21] and C₄₄Mab-46 [22,23]. We previously established an anti-CD44v4 mAb, C₄₄Mab-108 (mouse IgG₁, kappa) using the peptide immunization [19]. To clarify further characteristics of C₄₄Mab-108, we performed epitope mapping using enzyme-linked immunosorbent assay (ELISA).

2. Materials and Methods

2.1. Peptides

The CD44v4 peptide (₂₇₁-AFDHTKQNQDWTQWNPSHSN-₂₉₀) and 20 alanine (or glycine)-substituted peptides (Table 1) were synthesized by utilizing PEPScreen (Sigma-Aldrich Corp., St. Louis, MO). The number of amino acids (aa) is derived from human CD44v3-10 (accession No.: X66733).

2.2. ELISA

The CD44v4 peptides were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc., Waltham, MA) at a concentration of 10 µg/mL for 30 min at 37℃. After washing with phosphate-buffered saline (PBS) containing 0.05% Tween20 (PBST; Nacalai Tesque, Inc., Kyoto, Japan), wells were blocked with 1% bovine serum albumin (BSA)-containing PBST for 30 min at 37℃. The plates were incubated with 10 µg/mL of C₄₄Mab-108, followed by a peroxidase-conjugated anti-mouse immunoglobulins (1:2000 diluted; Agilent Technologies Inc., Santa Clara, CA). Enzymatic reactions were performed using the ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.). Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).

2.3. Flow cytometriy

The N-terminal PA16-tagged CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3-10) [19] cells were harvested after a brief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.) and washed with 0.1% BSA in PBS. C₄₄Mab-108 (10 μg/mL) was incubated with the CD44v4 peptides (10 μg/ml) for 30 min at 4°C. The cells were further treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000). Fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).

2.4. Immunohistochemical analysis

One formalin-fixed paraffin-embedded (FFPE) tissue of oral squamous cell carcinoma (OSCC) for peptide blocking assay was obtained from Tokyo Medical and Dental University [24]. The tissue slides were autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min for antigen retrieval. After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), the sections were incubated with C₄₄Mab-108 (10 μg/mL) in the presence or absence of the CD44v4 peptides (10 μg/mL) and then treated with the EnVision+ Kit for mouse (Agilent Technologies Inc.) for 30 min. The color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.). Counterstaining was performed with hematoxylin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany) was used to examine the sections and obtain images.

3. Results

3.1. Epitope mapping of C₄₄Mab-108 with alanine (or glycine)-substituted CD44v4 peptide

We previously established an anti-CD44v4 mAb (C₄₄Mab-108) by peptide immunization of CD44v4 region (_273-DHTKQNQDWTQWNPSHSNP_-291) [19]. We confirmed that C₄₄Mab-108 recognizes with only the variant 4-encoded region peptide (aa 271-290), but not other regions of CD44v3-10 extracellular domain [19]. To identify the binding epitope of C₄₄Mab-108, we synthesized 20 alanine (or glycine)-substituted peptides of the CD44v4 (Table 1). C₄₄Mab-108 exhibited reaction with A271G, F272A, D273A, H274A, T275A, K276A, Q277A, N278A, Q279A, T282A, Q283A, W284A, N285A, P286A, S287A, H288A, S289A, N290A, and wild-type (WT) (Figure 1A). In contrast, C₄₄Mab-108 did not react with D280A and W281A (Figure 1A). This result indicated that Asp280 and Trp281 are included in the critical epitope of C₄₄Mab-108. The results are summarized in Table 1. Figure 1B shows the schematic illustration of CD44s, CD44v3-10, and the critical aa (Asp280 and Trp281) recognized by C₄₄Mab-108.

3.2. Flow cytometry using C₄₄Mab-108 with alanine-substituted CD44v4 peptides

We next performed a peptide-blocking assay using flow cytometry to confirm the importance of the C₄₄Mab-108 epitope. As shown in Figure 2, C₄₄Mab-108 reacted with the CHO/CD44v3-10 cells. This reaction was completely neutralized by WT. In contrast, D280A and W281A did not block the reaction of C₄₄Mab-108 with CHO/CD44v3-10. The result confirmed that Asp280 and Trp281 of CD44v3-10 are critical for detection by C₄₄Mab-108 using flow cytometry.

3.3. Immunohistochemistry using C₄₄Mab-108 with alanine-substituted CD44v4 peptides

We also performed a peptide-blocking assay using immunohistochemical analysis. As shown in Figure 3, C₄₄Mab-108 stained the FFPE section of OSCC, which was completely neutralized by WT. In contrast, D280A and W281A did not neutralize the reaction. These results were corresponding to that of Figure 2.

4. Discussion

In the present study, the critical epitope of C₄₄Mab-108 was determined as Asp280 and Trp281 in the CD44v4 region. Since the reactivity of C₄₄Mab-108 to T282A and W284A was also reduced (Figure 1A), Thr282 and Trp284 may contribute to the recognition partially. Figure 1B shows the homology of the v4 region among human, mouse, Chinese hamster, and rat sequences. The Asp280 and Trp281 are conserved in human, mouse, Chinese hamster, but not in rat. Furthermore, Thr282 and Trp284 are also conserved in human and mouse. Although the result suggests that C₄₄Mab-108 might recognize both human and mouse CD44v4, it did not react with mouse CD44v4 using flow cytometry (data not shown).

Since C₄₄Mab-108 was established by the peptide immunization, C₄₄Mab-108 can recognize the definite peptide structure of the variant 4-encoded region. In contrast, CD44 is predicted to carry 146 O-glycan sites in the variant region. Among them, 41 of these sites have already been experimentally confirmed [25]. Thr282 is also a confirmed O-glycan site [26]. Further studies are required to reveal whether the O-glycan at Thr282 affects the recognition by C₄₄Mab-108.

In our previous study, C₄₄Mab-108 could detect CD44v3-10-overexpressed cells such as CHO/CD44v3-10, but not detect endogenous CD44v4 in several cancer cell lines in flow cytometry [19]. In contrast, C₄₄Mab-108 could detect endogenous and membranous CD44v4 in immunohistochemistry [19]. These results suggest that the variant 4-encoded region is folded into the inside of protein in living cells, but exposed by antigen retrieval in immunohistochemistry. Recently, we provided a potential strategy for developing cancer-specific antibodies that target locally misfolded cell surface receptors such as human epidermal growth factor receptor 2 [27,28]. Further studies are needed to investigate whether C₄₄Mab-108 is involved in the recognition of specific CD44v4 type and/or specific condition of cells.