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

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Abstract
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. The results demonstrated that Tyr11, Tyr12, Glu14, Glu15, and Glu17 are critical for recognition by A4Mab-1, while Tyr11, Tyr12, Tyr13, Glu15, and Asn16 are essential for recognition by A4Mab-2 in flow cytometry and Western blotting. Furthermore, Glu14, Asn16, and Glu17 are essential for recognition by A4Mab-3 in flow cytometry. These findings contribute to the understanding of mACKR4 recognition by A4Mabs.

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