Establishment of Novel Anti-EphB3 Monoclonal Antibodies for Multiple Applications

Ephrin type-B receptor 3 (EphB3) binds to transmembrane ephrin-B ligands to regulate cell migration, adhesion, and proliferation. EphB3 exhibits a gradient expression pattern in the normal intestine, with the highest levels at the crypt base, and plays a crucial role in the maintenance of normal intestinal epithelium. Therefore, anti-EphB3 monoclonal antibodies (mAbs) are required for basic research and diagnosis. In this study, we developed novel antihuman EphB3, Eb₃Mab-5 (IgG₁, κ) and Eb₃Mab-11 (IgG₁, κ), using the Cell-Based Immunization and Screening (CBIS) method. Eb₃Mab-5 and Eb₃Mab-11 reacted with EphB3-overexpressed Chinese hamster ovary-K1 (CHO/EphB3) and endogenous EphB3-positive colorectal cancer LS174T in flow cytometry. The apparent binding affinity of Eb₃Mab-5 for CHO/EphB3 and LS174T was 7.6 × 10^-9 M and 1.7 × 10^-8 M, respectively. Eb₃Mab-11 could detect EphB3 in western blot analysis and immunohistochemistry. Eb₃Mab-5 and Eb₃Mab-11, established by the CBIS method, may contribute to the diagnosis and therapy of EphB3-positive tumors.