Epitope analysis of an anti-mouse CCR1 monoclonal antibody S15040E using flow cytometry

Ayaka Okada; Hiroyuki Suzuki; Takao Arimori; Tomohiro Tanaka; Mika K Kaneko; Yukinari Kato

doi:10.1016/j.bbrep.2025.102265

Epitope analysis of an anti-mouse CCR1 monoclonal antibody S15040E using flow cytometry

Biochem Biophys Rep. 2025 Sep 13:44:102265. doi: 10.1016/j.bbrep.2025.102265. eCollection 2025 Dec.

Authors

Ayaka Okada¹, Hiroyuki Suzuki¹, Takao Arimori², Tomohiro Tanaka¹, Mika K Kaneko¹, Yukinari Kato¹

Affiliations

¹ Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
² Institute for Protein Research, Osaka University, 3-2. Yamadaoka, Suita, Osaka, 565-0871, Japan.

Abstract

The C-C motif chemokine receptor 1 (CCR1) is widely expressed in various immune cells and plays crucial roles in the maturation and migration of immune cells. CCR1 has been considered an attractive drug target for treating autoimmune diseases and tumors. An anti-mouse CCR1 (mCCR1) monoclonal antibody (clone S15040E) has been used in various in vivo studies to identify mCCR1-positive cells by flow cytometry. However, the binding epitope has not been determined. This study investigated the binding epitope of S15040E using flow cytometry. The mCCR1 extracellular domain-substituted mutant analysis showed that S15040E recognizes the extracellular loop 2 (ECL2, aa 172-197) of mCCR1. Next, alanine (or glycine) scanning was conducted in the ECL2 region. The results revealed that Trp176, Phe178, and Arg181 are essential amino acids for the recognition by S15040E. These results showed the involvement of the ECL2 of mCCR1 in the recognition by S15040E.

Keywords: Alanine scanning; Epitope mapping; Flow cytometry; Monoclonal antibody; Mouse CCR1.