Development of specific anti-mouse atypical chemokine receptor 4 monoclonal antibodies.

Leukocyte migration is an essential function of innate and adaptive immune responses. Chemokines and their receptors control the migration system. The abundance of chemokines is controlled by atypical chemokine receptors (ACKRs), chemokine receptor-like molecules that do not couple to the G protein signaling pathways. Among them, ACKR4 regulates dendritic cell migration by controlling the ligands and is involved in tumor development in mouse models. Because no anti-mouse ACKR4 (mACKR4) monoclonal antibody (mAb) for flow cytometry has been reported, this study aimed to develop a novel mAb for mACKR4. Among the established anti-mACKR4 mAbs, AMab-1 (rat IgG, kappa), AMab-2 (rat IgG, kappa), and AMab-3 (rat IgG, kappa) recognized mACKR4-overexpressed Chinese hamster ovary-K1 (CHO/mACKR4) by flow cytometry. The dissociation constant ( ) values of AMab-1, AMab-2, and AMab-3 for CHO/mACKR4 were determined as 6.0 × 10 M, 1.3 × 10 M, and 1.7 × 10 M, respectively. Furthermore, AMab-1 and AMab-2 could detect mACKR4 by western blotting. These results indicated that AMab-1, AMab-2, and AMab-3 help to detect mACKR4 by flow cytometry and western blotting and obtain the proof of concept in preclinical models.