Development of a Novel Anti-CD44 Variant 6 Monoclonal Antibody C₄₄Mab-9 for Multiple Applications against Colorectal...

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C₄₄Mab-9; IgG₁, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C₄₄Mab-9 recognizes CD44v6. Furthermore, C₄₄Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (K_D) of C₄₄Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10^-9 M, 1.7 × 10^-8 M, and 2.3 × 10^-8 M, respectively. C₄₄Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C₄₄Mab-9 is useful for detecting CD44v6 in various applications.