RESUMEN
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, A4Mab-1 (rat IgG2b, kappa), A4Mab-2 (rat IgG2b, kappa), and A4Mab-3 (rat IgG2b, kappa) recognized mACKR4-overexpressed Chinese hamster ovary-K1 (CHO/mACKR4) by flow cytometry. The dissociation constant (K D) values of A4Mab-1, A4Mab-2, and A4Mab-3 for CHO/mACKR4 were determined as 6.0 × 10-9 M, 1.3 × 10-8 M, and 1.7 × 10-9 M, respectively. Furthermore, A4Mab-1 and A4Mab-2 could detect mACKR4 by western blotting. These results indicated that A4Mab-1, A4Mab-2, and A4Mab-3 help to detect mACKR4 by flow cytometry and western blotting and obtain the proof of concept in preclinical models.
RESUMEN
One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands, CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of proinflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8 for CHO/mCCR5 were determined as 3.5 × 10-8 M and 7.3 × 10-9 M, respectively. Furthermore, both C5Mab-4 and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies.