A Novel Anti-Cadherin 19 Monoclonal Antibody (Ca19Mab-8) for Flow Cytometry, Western Blotting, and Immunohistochemistry

1. Introduction

Cadherin-19 (CDH19) is a neural crest–specific adhesion molecule that plays a central role in maintaining brain homeostasis [1,2]. CDH19 belongs to the classical type II cadherin family and comprises five extracellular cadherin repeats (EC1–EC5), a single-pass transmembrane region, and a cytoplasmic tail [3]. The extracellular domain of CDH19 mediates calcium-dependent homophilic interactions at adherens junctions [4]. Its cytoplasmic domain associates with α-catenin, β-catenin, and p120-catenin, which link CDH19 to the actin cytoskeleton [5,6].

The human CDH19 gene was cloned in 2000 based on its sequence similarity to CDH7 [7]. Expressed sequence tags for CDH19 were isolated from melanocyte cDNA libraries, suggesting that CDH19 expression may be restricted to neural crest-derived cells [7]. Supporting this observation, rat CDH19 was primarily expressed in nerve ganglia and Schwann cells during embryonic development. Moreover, CDH19 expression overlapped with neural crest markers such as AP-2 and Sox10 [2]. In Sox10-knockout mouse embryos, neural crest cells exhibited delayed migration to the distal hindgut along with a simultaneous downregulation of CDH19 [1]. Furthermore, Sox10 was found to bind to the CDH19 promoter, indicating that CDH19 is a direct target of Sox10 during early neural crest cell migration by forming CDH19-catenins-cytoskeleton complexes [1]. Through these functions, CDH19 is crucial for the development of neural crest cells, providing insights into the pathogenesis of nervous system developmental defects [8,9].

Melanoma is a type of skin cancer caused by the oncogenic transformation of melanocytes, which are pigment-producing skin cells derived from the neural crest [10,11]. Estimated new cases of melanoma in 2025 are more than 100,000 in the US [12], and approximately 20% of patients have advanced disease [American Joint Committee on Cancer (AJCC) stages: IIIA~IIID] at the time of diagnosis [13]. Although primary melanoma can be cured by surgery, and immune checkpoint inhibitors are the standard care in patients with advanced-stage melanoma [14,15], melanoma is the leading cause of death from skin disease in the US, responsible for 8,430 estimated deaths in 2025 [12]. A single-cell multi-omics analysis of human melanoma revealed that melanoma cells were grouped into seven subtypes, which include a CDH19-high subtype, which was suggested to be more resistant to NK and T cell-mediated immunity [16].

Monoclonal antibodies (mAbs) that detect CDH19 by Western blotting or immunohistochemistry (IHC) have been developed for various applications. However, no mAb is available for flow cytometry. Using the Cell-Based Immunization and Screening (CBIS) method, anti-E-Cadherin/CDH1 [17] and anti-M-Cadherin/CDH15 [18] mAbs for flow cytometry, Western blotting, and IHC were developed by our laboratory. The CBIS method includes high-throughput flow cytometry–based screening. Therefore, mAbs obtained by the CBIS method generally recognize conformational epitopes, enabling their use in flow cytometry. Some of the mAbs are suitable for Western blotting and IHC. In this study, we employed the CBIS method to develop highly versatile anti-CDH19 mAbs.

2. Materials and Methods

2.1. Cell Lines

Chinese hamster ovary (CHO)-K1, mouse myeloma P3X63Ag8U.1 (P3U1), and human glioblastoma (GBM) LN229 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

2.2. Plasmid Construction and Establishment of Stable Transfectants

Genes encoding human CDH19 (NM_021153) were purchased from OriGene Technologies, Inc. (Rockville, MD, USA). The CDH19 cDNA without the pro-peptide was subcloned into the pCAG-Ble vector with an N-terminal PA16 tag [19]. Additionally, the CDH19 cDNA with an N-terminal MAP16 tag [20] was constructed. These plasmids were transfected into CHO-K1 or LN229 cells, and stable transfectants were sorted using anti-PA16 tag mAb (clone NZ-1) [19] or anti-MAP16 tag mAb (clone PMab-1) [20] using the Neon transfection system (Thermo Fisher Scientific, Inc.). Finally, PA16-CDH19-overexpressed CHO-K1 (CHO/CDH19) and MAP16-CDH19-overexpressed LN229 (LN229/CDH19) were established.

We previously established other CDH-overexpressed stable transfectants [21]. To confirm the expression of CDHs in these transfectants, 1 μg/mL of an anti-CDH1 mAb (clone 67A4), 1 μg/mL of an anti-CDH3 mAb (clone MM0508-9V11), or 0.1 μg/mL of an anti-PA16 tag mAb, NZ-33 [22] were used.

2.3. Production of Hybridomas

Female BALB/cAJcl mice (CLEA Japan, Tokyo, Japan) were intraperitoneally immunized with LN229/CDH19 cells (1 × 10⁸ cells/injection) mixed with 2% Alhydrogel adjuvant (InvivoGen, San Diego, CA, USA). Following three additional weekly immunizations (1.0 × 10⁸ cells/injection), a booster dose (1 × 10⁸ cells/injection) was administered two days before spleen excision. Hybridomas were produced as previously described [18].

2.4. Flow Cytometry

A total of 1 × 10⁵ cells harvested with 1 mM EDTA were washed with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA; blocking buffer) and incubated with mAbs for 30 minutes at 4°C. Flow cytometric data were acquired as described previously [17].

2.5. Determination of Dissociation Constant Values Using Flow Cytometry

LN229/CDH19 cells were treated with serially diluted Ca₁₉Mab-8. The dissociation constant (K_D) values were calculated s described previously [17].

2.6. Western Blotting

Western blotting was conducted using 1 μg/mL Ca₁₉Mab-8 or 1 μg/mL of an anti-isocitrate dehydrogenase 1 (IDH1) mAb (clone RcMab-1) as described previously [18].

2.7. IHC Using Cell Blocks

The formalin-fixed paraffin-embedded (FFPE) cell sections were prepared as described previously [18].They are stained with 0.2 μg/mL of Ca₁₉Mab-8 or 0.01 μg/mL of NZ-33 using the BenchMark ULTRA PLUS with the ultraView Universal DAB Detection Kit (Roche Diagnostics, Indianapolis, IN, USA).

3. Results

3.1. Anti-CDH19 mAb Development by the CBIS Method

As described in the Materials and Methods, an immunogen, LN229/CDH19, was prepared. LN229/CDH19 (1 × 10⁸ cells/mouse) was immunized five times into two BALB/cAJcl mice (Figure 1A). Subsequently, hybridomas were generated by fusing splenocytes with myeloma P3U1 (Figure 1B). The hybridoma supernatants were screened to identify those positive for CHO/CDH19 and negative for CHO-K1 (Figure 1C). As a result, 43 positive wells out of 958 (4.5%) were found. Limiting dilution was then performed to clone hybridomas producing anti-CDH19 mAb (Figure 1D). Ultimately, 12 clones were established (http://www.med-tohoku-antibody.com/topics/001_paper_antibody_PDIS.htm#CDH19+), and the purified mAbs (IgG₁ isotype) were prepared.

3.2. Determination of the Specificity of Ca₁₉Mab-8 Using CDHs-Overexpressed CHO-K1

We previously established CHO-K1 cells, which overexpressed type I CDHs (CDH1, CDH2, CDH3, CDH4, and CDH15) [17,18], type II CDHs (CDH5, CDH6, CDH7, CDH8, CDH9, CDH10, CDH11, CDH12, CDH18, CDH19, CDH20, CDH22, and CDH24), 7D CDHs (CDH16 and CDH17), a truncated CDH (CDH13), and an atypical CDH (CDH26) [21]. Therefore, the specificity of Ca₁₉Mabs to those CDHs was determined. As shown in Figure 2A, Ca₁₉Mab-8 reacted with CHO/CDH19 but did not react with other CDHs-overexpressed in CHO-K1. The cell surface expression of CDHs was confirmed in Figure 2B. In contrast, other Ca₁₉Mabs showed cross-reactivity against other type II CDHs (Supplementary Table S1). These results indicate that Ca₁₉Mab-8 is a specific mAb to CDH19 among those CDHs.

3.3. Flow Cytometry of Ca₁₉Mab-8 Against CDH19-Overexpressed CHO-K1 and LN229

We next conducted flow cytometry using Ca₁₉Mab-8 (IgG₁, κ) against CHO/CDH19, CHO-K1, LN229/CDH19, and LN229. Ca₁₉Mab-8 reacted with CHO/CDH19 in a dose-dependent manner, from 10 to 0.01 μg/mL (Figure 3A). In contrast, Ca₁₉Mab-8 did not recognize CHO-K1 at 10 μg/mL (Figure 3A). Furthermore, Ca₁₉Mab-8 reacted with LN229/CDH19 in a dose-dependent manner (Figure 3A). Ca₁₉Mab-8 also showed reactivity to parental LN229, suggesting that LN229 expressed endogenous CDH19. The binding affinity of Ca₁₉Mab-8 was measured using flow cytometry. The fitting binding isotherms of Ca₁₉Mab-8 to LN229/CDH19 are shown in Supplementary Figure 1. The K_D value of Ca₁₉Mab-8 for LN229/CDH19 was 9.0 × 10⁻⁹ M. These results indicate that Ca₁₉Mab-8 possesses a moderate binding affinity to LN229/CDH19.

3.4. Western Blotting Using Ca₁₉Mab-8

We then examined whether Ca₁₉Mab-8 is suitable for Western blotting. Whole-cell lysates from CHO-K1, CHO/CDH19, LN229, and LN229/CDH19 were analyzed. Ca₁₉Mab-8 detected clear bands around 90–100 kDa in CHO/CDH19 and LN229/CDH19, but not in CHO-K1 (Figure 4A). It also detected weak bands in parental LN229 (Figure 4A). Figure 4B shows an internal control, IDH1, detected by RcMab-1. These results demonstrate that Ca₁₉Mab-8 can detect both exogenous and endogenous CDH19 in Western blotting.

3.5. IHC using Ca₁₉Mab-8 in Formalin-Fixed Paraffin-Embedded Cell Blocks

We assessed whether Ca₁₉Mab-8 is suitable for immunohistochemistry (IHC) of FFPE sections from CHO-K1 and CHO/CDH19. Ca₁₉Mab-8 showed strong cytoplasmic and membranous staining in CHO/CDH19 but not in CHO-K1 (Figure 5A). Additionally, an anti-PA16 tag mAb, NZ-33, exhibited cytoplasmic and membranous staining in CHO/CDH19, but not in CHO-K1 (Figure 5B). These results suggest that Ca₁₉Mab-8 can detect CDH19 in IHC of FFPE sections of cultured cells.

4. Discussion

This study identified novel anti-CDH19 mAbs using the CBIS method (Figure 1). Among them, Ca₁₉Mab-8 recognized both exogenous and endogenous CDH19 in flow cytometry and Western blotting (Figure 3 and Figure 4). Ca₁₉Mab-8 specifically binds to CDH19 but not to other type II, type I, 7D, truncated, or atypical CDHs (Figure 2). In contrast, the other nine Ca₁₉Mabs showed cross-reactivity with CDH7 and other type II CDHs in flow cytometry (Supplementary Table S1). Since most commercially available mAbs lack information on cross-reactivity, careful handling and interpretation are essential when using them. Identifying the Ca₁₉Mab-8 epitope is crucial for developing more specific anti-CDH19 mAbs. Additionally, Ca₁₉Mab-8 is suitable for IHC on cell blocks (Figure 5). IHC was performed using an automated slide-staining system, allowing for standardized staining conditions. Ca₁₉Mab-8 is highly versatile for both basic research and clinical applications.

We demonstrated that Ca₁₉Mab-8 recognized human GBM LN229 cells in flow cytometry and Western blotting (Figure 3 and Figure 4). Although we examined the reactivity of Ca₁₉Mab-8 in other glioblastoma cell lines, LN229 is the only cell line identified by Ca₁₉Mab-8. In contrast, CDH19 was detected by Western blotting in GBM stem-like cells isolated from fresh GBM samples [23]. We should investigate the reactivity of Ca₁₉Mab-8 with those samples and assess its antitumor efficacy. We previously cloned cDNA of mAb and produced recombinant mouse IgG_2a-type mAbs to confer antibody-dependent cellular cytotoxicity (ADCC). These mAbs have been evaluated for the antitumor efficacy in human tumor xenograft models [24,25]. We have cloned the cDNA of Ca₁₉Mab-8, and recombinant Ca₁₉Mab-8 will be produced and evaluated for in vitro ADCC activity and antitumor activity in mouse glioblastoma xenograft models.

Metastatic melanoma remains a major clinical challenge [26,27]. Although therapeutic outcomes have significantly improved since the introduction of immune checkpoint inhibitors, about half of patients with metastatic melanoma do not achieve long-term survival benefits [28,29]. A recent single-cell and spatial multi-omics analysis showed that drug-naive human melanoma biopsies contain cancer cells in a mesenchymal-like (MES) state [30]. Melanoma cells in the MES state exhibit resistance to targeted therapy and immunotherapy and are more frequently found in lesions that do not respond to treatment [30].

CDH19 was identified as a MES signature regulated by the master transcription factor TCF4. Targeting TCF4 genetically enhances immunogenicity and increases the sensitivity of MES cells to immunotherapy and targeted treatments [30]. Therefore, CDH19 is a promising antigen for targeting drug-resistant melanoma cells in the MES state. Moreover, a patent (US 2025/0084160 A1, Mar.13, 2025) reports that CDH19 is expressed in melanoma cell lines, such as CHL-1, which can serve as a preclinical model for melanoma therapy. Since Ca₁₉Mab-8 can detect CDH19-positive cells by flow cytometry without cross-reactivity (Figure 2), Ca₁₉Mab-8 will be a key foundation for developing various modalities, including antibody-drug conjugates, bispecific antibodies, and chimeric antigen receptor T cells.