Development of Sensitive Anti-Mouse CCR5 Monoclonal Antibodies Using the N-Terminal Peptide Immunization.

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, C ₅ Mab-4 (rat IgG _2a , kappa) and C ₅ Mab-8 (rat IgG ₁ , kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant ( K _D ) values of C ₅ Mab-4 and C ₅ Mab-8 for CHO/mCCR5 were determined as 3.5 × 10 ^-8 M and 7.3 × 10 ^-9 M, respectively. Furthermore, both C ₅ Mab-4 and C ₅ Mab-8 could detect mCCR5 by western blotting. These results indicated that C ₅ Mab-4 and C ₅ Mab-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.