Epitope Mapping of Anti-mouse ACKR4 Monoclonal Antibodies Developed by N-Terminal Peptide Immunization

Leukocyte migration is a fundamental process in both innate and adaptive immune responses. This process is tightly regulated by chemokines and their cognate receptors. The bioavailability of chemokines is further modulated by atypical chemokine receptors (ACKRs), a subset of chemokine receptor-like molecules that lack coupling to canonical G protein-mediated signaling pathways. Among these, ACKR4 regulates dendritic cell migration through ligand scavenging and has been implicated in tumor progression in murine models. We previously established anti-mouse ACKR4 (mACKR4) mAbs, A₄Mab-1, A₄Mab-2, and A₄Mab-3, by N-terminal peptide immunization. This study examined the binding epitopes of A₄Mabs. Alanine (or glycine) scanning within the N-terminal region (amino acids 2-19) was performed using flow cytometry and Western blotting. Results showed that Tyr12 is required for recognition by A₄Mab-1 in flow cytometry, whereas Tyr11, Tyr12, Glu14, Glu15, and Glu17 are required in Western blotting. For A₄Mab-2, Tyr12, Glu15, and Asn16 are required in flow cytometry, whereas Tyr11, Tyr12, Tyr13, Glu15, and Asn16 are required in Western blotting. Additionally, Glu14, Asn16, and Glu17 are required for recognition by A₄Mab-3 in flow cytometry. These findings contribute to the understanding of mACKR4 recognition by A₄Mabs.