Ca₁₆Mab-56, a Novel Anti-Cadherin-16/Ksp-Cadherin Monoclonal Antibody for Multiple Applications

Background/Objectives: Cadherin-16 (CDH16, Ksp-cadherin) possesses unique seven extracellular cadherin repeats, and its expression is restricted to normal kidney epithelium. CDH16 is downregulated in renal cell carcinoma (RCC) and is associated with poor prognosis. Therefore, developing mAbs that specifically recognize cell-surface CDH16 is essential for tumor diagnosis and for isolating CDH16-positive renal epithelial cells. Methods: Anti-human CDH16 mAbs (designated as Ca₁₆Mabs) were developed by immunizing mice with CDH16-overexpressed tumor cells, followed by a high-throughput flow cytometry-based screening. Results: Among the 58 established Ca₁₆Mabs, a clone, Ca₁₆Mab-56 (IgG₁, κ), specifically recognized CDH16-overexpressed Chinese hamster ovary-K1 (CHO/CDH16) cells with no detectable cross-reactivity to 21 other CDHs in flow cytometry. Ca₁₆Mab-56 also detected endogenous CDH16 in human RCC cell lines (OS-RC-2 and KMRC-20) and normal kidney epithelial cell lines. The dissociation constant (K_D) values of Ca₁₆Mab-56 for CHO/CDH16 and OS-RC-2 were determined as 6.5 × 10⁻⁹ M and 1.2 × 10⁻⁹ M, respectively. Furthermore, Ca₁₆Mab-56 detected endogenous CDH16 by Western blotting and showed potent staining in normal kidney tubular epithelium and clear membranous staining in renal cell carcinoma in immunohistochemistry. Conclusion: Ca₁₆Mab-56 is a versatile tool for detecting CDH16 and has potential for tumor diagnosis.