Establishment of a Novel Anti-CD44 Variant 10 Monoclonal Antibody C44Mab-18 for Immunohistochemical Analysis against Oral Squamous Cell Carcinomas

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44 has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer diagnosis and therapy. We developed monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed PANC-1 cells. Among the established clones, C44Mab-18 (IgM, kappa) reacted with CHO/CD44v3–10, but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping using peptides that cover variant exon-encoded regions revealed that C44Mab-18 recognized the border sequence between variant 10 and the constant exon 16-encoded sequence. These results suggest that C44Mab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore, C44Mab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow cytometry. The apparent dissociation constant (KD) of C44Mab-18 for CHO/CD44v3–10 and HSC-3 was 1.6 × 10−7 M and 1.7 × 10−7 M, respectively. Furthermore, C44Mab-18 detected CD44v3–10 but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry using OSCC tissues. These results indicate that C44Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.