Citation: Tanaka, T.; Tawara, M.; Suzuki, H.; Kaneko, M.K.; Kato, Y. Identification of the Binding Epitope of an Anti-Mouse CCR6 Monoclonal Antibody (C 6 Mab-13) Using 1× Alanine Scanning. Antibodies 2023, 12, 32. https://doi.org/10.3390/ antib12020032 Academic Editor: Anthony Rees Received: 16 March 2023 Revised: 28 March 2023 Accepted: 17 April 2023 Published: 28 April 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antibodies Communication Identification of the Binding Epitope of an Anti-Mouse CCR6 Monoclonal Antibody (C 6 Mab-13) Using 1× Alanine Scanning Tomohiro Tanaka 1 , Mayuki Tawara 1 , Hiroyuki Suzuki 1, * , Mika K. Kaneko 1,2 and Yukinari Kato 1,2, * 1 Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; tomohiro.tanaka.b5@tohoku.ac.jp (T.T.); tawara.mayuki.p8@dc.tohoku.ac.jp (M.T.); k.mika@med.tohoku.ac.jp (M.K.K.) 2 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan * Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.); Tel.: +81-22-717-8207 (H.S. & Y.K.) Abstract: CC chemokine receptor 6 (CCR6) is one of the members of the G-protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, such as B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. The coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, psoriasis, and autoimmune diseases. Thus, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for various diseases. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C 6 Mab-13 (rat IgG 1 , kappa), that was applicable for flow cytometry by immunizing a rat with the N-terminal peptide of mCCR6. In this study, we investigated the binding epitope of C 6 Mab-13 using an enzyme- linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) method, which were conducted with respect to the synthesized point-mutated-peptides within the 1–20 amino acid region of mCCR6. In the ELISA results, C 6 Mab-13 lost its ability to react to the alanine-substituted peptide of mCCR6 at Asp11, thereby identifying Asp11 as the epitope of C 6 Mab-13. In our SPR analysis, the dissociation constants (K D ) could not be calculated for the G9A and D11A mutants due to the lack of binding. The SPR analysis demonstrated that the C 6 Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C 6 Mab-13 was determined to be located around Asp11 on mCCR6. Based on the epitope information, C 6 Mab-13 could be useful for further functional analysis of mCCR6 in future studies. Keywords: mouse CCR6; monoclonal antibody; epitope; ELISA; SPR 1. Introduction The CC chemokine receptor 6 (CCR6) is a seven-transmembrane chemokine receptor belonging to the G-protein-coupled receptor (GPCR) family [1–5]. CCR6 was identified as a specific receptor for the CC motif chemokine ligand 20 (CCL20) in 1997 [6]. It is report- edly associated with various diseases, such as cancer [7–9], autoimmune diseases [10–13], psoriasis [14–17], and inflammatory bowel disease (IBD) [18–22]. The expression of CCR6 is found in B cells or T cells [23], such as effector memory T cells [24], immature dendritic cells [25], Th17 cells [26], and regulatory T (Treg) cells [27], and thus affects the activity and directionality of immune cells [23,24,26]. Mice lacking CCR6 exhibited impaired leukocyte homeostasis, which results in severe contact hypersensitivity and defects in delayed-type hypersensitivity responses. These results suggested that CCR6 plays a critical role in the regulation of leukocyte homeostasis [28]. The chemokine ligand CCL20, also known as macrophage inflammatory protein-3α (MIP-3α)[29], liver- and activation-regulated chemokine (LARC) [30], or Exodus-1 [31], is a crucial CCR6 ligand. The binding of CCL20 to CCR6 can activate a variety of intracellular Antibodies 2023, 12, 32. https://doi.org/10.3390/antib12020032 https://www.mdpi.com/journal/antibodies