Establishment of a Novel Anti-Human CCR6 Monoclonal Antibody C6Mab-19 with the High Binding Affinity in Flow Cytometry.

Abstract

CC chemokine receptor 6 (CCR6) is a member of the G-protein-coupled receptor family that is highly expressed in B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. CCR6 has been revealed to have important functions in many pathological conditions, such as cancer, intestinal bowel disease, psoriasis, and autoimmune diseases. The only CCR6 chemokine ligand, CC motif chemokine ligand 20 (CCL20), is also involved in pathogenesis by interacting with CCR6. The CCL20/CCR6 axis is drawing attention as an attractive therapeutic target for various diseases. In this study, we developed novel monoclonal antibodies (mAbs) against human CCR6 (hCCR6) using the peptide immunization method, which are applicable to flow cytometry and immunohistochemistry. The established anti-hCCR6 mAb, clone C₆Mab-19 (mouse IgG₁, kappa), reacted with hCCR6-overexpressed Chinese hamster ovary-K1 (CHO/hCCR6), human liver carcinoma (HepG2), and human differentiated hepatoma (HuH-7) cells in flow cytometry. The dissociation constant (K_D) of C₆Mab-19 was determined as 3.0 × 10^-10 M for CHO/hCCR6, 6.9 × 10^-10 M for HepG2, and 1.8 × 10^-10 M for HuH-7. Thus, C₆Mab-19 could bind to exogenously and endogenously expressed hCCR6 with extremely high affinity. Furthermore, C₆Mab-19 could stain formalin-fixed paraffin-embedded lymph node tissues from a patient with non-Hodgkin lymphoma by immunohistochemistry. Therefore, C₆Mab-19 is suitable for detecting hCCR6-expressing cells and tissues and could be useful for pathological analysis and diagnosis.