Development of a Sensitive Anti-Human CCR9 Monoclonal Antibody (C9Mab-11) by N-Terminal Peptide Immunization.

Abstract

The C-C chemokine receptor 9 (CCR9) belongs to the G-protein-coupled receptor superfamily, and is highly expressed on the T cells and intestinal cells. CCR9 regulates various immune responses by binding to the C-C chemokine ligand, CCL25, and is involved in inflammatory diseases and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR9 is necessary for treatment and diagnosis. In this study, we established a specific anti-human CCR9 (hCCR9) mAb; C₉Mab-11 (mouse IgG_2a, kappa), using the synthetic peptide immunization method. C₉Mab-11 reacted with hCCR9-overexpressed Chinese hamster ovary-K1 (CHO/hCCR9) and hCCR9-endogenously expressed MOLT-4 (human T-lymphoblastic leukemia) cells in flow cytometry. The dissociation constant (K_D) of C₉Mab-11 for CHO/hCCR9 and MOLT-4 cells were determined to be 1.2 × 10^-9 M and 4.9 × 10^-10 M, respectively, indicating that C₉Mab-11 possesses a high affinity for both exogenously and endogenously hCCR9-expressing cells. Furthermore, C₉Mab-11 clearly detected hCCR9 protein in CHO/hCCR9 cells using western blot analysis. In summary, C₉Mab-11 can be a useful tool for analyzing hCCR9-related biological responses.