Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody, C₄₄Mab-34, for Multiple Applications against Oral...

Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3-10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C₄₄Mab-34; IgG₁, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that C₄₄Mab-34 is a specific mAb for CD44v7/8. Moreover, C₄₄Mab-34 reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent K_D of C₄₄Mab-34 for CHO/CD44v3-10 and HSC-3 was 1.4 × 10^-9 and 3.2 × 10^-9 M, respectively. C₄₄Mab-34 could detect CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C₄₄Mab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.