Epitope Mapping of an Anti-Mouse CCR2 Monoclonal Antibody (C 2 Mab-6) Using Enzyme-Linked Immunosorbent Assay

Introduction

Chemokine receptors are G protein-coupled receptor (GPCR) with seven transmembrane regions, which are localized on the plasma membrane. Chemokines are divided into four different subfamilies of XC, CC, CXC, and CX3C, according to the number and position of conserved N-terminus cysteine residues. ¹ Chemokines orchestrate the cellular function of immune cells through their interaction with their receptors, as represented by cell migration. ²

CC chemokine receptor type-2 (CCR2) is expressed in various immune cells including T lymphocytes, natural killer cells, macrophages, dendritic cells, and monocytes. CCR2 is primarily involved in the regulation of migration and positioning of immune cells.^3,4 CCR2 is the major receptor of C-C motif chemokine 2 (CCL2)/monocyte chemoattractant protein-1. CCL2–CCR2 axis plays pivotal roles in the regulation of immune system. ⁵

At the inflammation site, CCR2-expressing cells are associated with tissue damage. During influenza A virus infection, interferon-γ-stimulated CCR2⁺ monocytes infiltration has been reported as a driver of lung damage. ⁶ High levels of CCR2 in peripheral blood have been detected in severe COVID-19 patients. ⁷ Genome-wide studies suggest that 3p21.31 is involved in the risk of severe COVID-19 aggravation and is involved in upregulation of chemokine receptors, including CCR2 in monocytes and macrophages. ⁸

CCL2 expression has been reported to be upregulated in several tumors, including breast, bladder, and bone cancers.^9–11 High CCR2 expression in tumor-infiltrating immune and stromal cells has been confirmed,^12–14 and associated with poor prognosis.^15,16 Therefore, the CCL2–CCR2 axis is thought to be an important target for cancer therapy.

We have developed monoclonal antibodies (mAbs) against chemokine receptors, including mouse CCR2 (mCCR2), ¹⁷ mouse CCR3, ¹⁸ mouse CCR4, ¹⁹ mouse CCR8, ²⁰ human CCR9, ²¹ and mouse CXCR6 ²² by the Cell-Based Immunization and Screening method or the N-terminal peptide immunization method. In this study, we investigated the epitope of an anti-mCCR2 mAb, C₂Mab-6, ¹⁷ by using enzyme-linked immunosorbent assay (ELISA).

Materials and Methods

Results

Epitope identification using N-terminal mCCR2 peptides

An anti-mCCR2 mAb, C₂Mab-6 (rat IgG₁, kappa), was established by immunization with the mixture of three keyhole limpet hemocyanin-conjugated mCCR2 N-terminal peptides. ¹⁷ The sequences are listed in Table 1. To characterize the binding epitope of C₂Mab-6, we first investigated the reactivity of C₂Mab-6 to the N-terminal synthetic peptides: 1–19 amino acids (aa), 11–29 aa, and 21–39 aa of mCCR2 (Table 1). The results of ELISA demonstrated that C₂Mab-6 recognized 1–19 aa of mCCR2 (Fig. 1A). These results are summarized in Figure 1B.

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FIG. 1.

Identification of the C₂Mab-6 epitope for mCCR2 by ELISA using N-terminal peptides. (A) N-terminal synthesized peptides of mCCR2 were immobilized on immunoplates. The plates were incubated with C₂Mab-6 (1 μg/mL) followed by incubation with peroxidase-conjugated anti-rat immunoglobulins. (B) Schematic illustration of mCCR2 and the C₂Mab-6 epitope. ELISA, enzyme-linked immunosorbent assay; mCCR2, mouse CCR2.

Epitope identification using alanine-substituted mCCR2 peptides

We further synthesized 20 different alanine-substituted mCCR2 peptides (Table 2). The results of ELISA demonstrated that C₂Mab-6 bound to point mutants, such as M1A, E2A, N5A, L7A, I11A, H12A, G13A, I14A, L15A, S16A, T17A, S18A, H19A, and S20A as well as the 1–20 aa wild type (WT) sequence (positive control) (Fig. 2A). In contrast, C₂Mab-6 did not bind to point mutants, such as the D3A, N4A, M6A, P8A, Q9A, and F10A (Fig. 2A), indicating that Asp3, Asn4, Met6, Pro8, Gln9, and Phe10 were determined to be the critical aa, included in the C₂Mab-6 epitope. The results are summarized in Figure 2B.

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FIG. 2.

Identification of the C₂Mab-6 epitope for mCCR2 by ELISA using alanine-substituted peptides. (A) Synthesized peptides of mCCR2 were immobilized on immunoplates. The plates were incubated with C₂Mab-6 (1 μg/mL), followed by peroxidase-conjugated anti-rat immunoglobulins. (B) Schematic illustration of mCCR2 and the C₂Mab-6 epitope. The C₂Mab-6 epitope of mCCR2 involves Asp3, Asn4, Met6, Pro8, Gln9, and Phe10.

Discussion

We previously developed an anti-human CCR2 mAb (clone C₂Mab-9) by using N-terminal peptide immunization method and determined the epitope of C₂Mab-9 as Phe23, Phe24, Asp25, and Tyr26. ²³ In this study, we successfully determined that the epitope of an anti-mCCR2 mAb (clone C₂Mab-6) ¹⁷ as Asp3, Asn4, Met6, Pro8, Gln9, and Phe10 (Fig. 2). The N-terminal regions of some GPCRs, including CCR2, CCR3, CCR5, and CXCR1, have been identified as their ligand-binding sites. ¹ Recently, cryoelectron microscopy structure of human CCL2 bound to CCR2-G-protein complex was reported.

The structural analysis reveals that N-terminal CCL2 deeply inserts into the extracellular half of the CCR2 transmembrane domain, and forms substantial interactions with CCR2 (Cys113, Thr117, Tyr120, His121, Cys190, Gly191, and P192) through the most N-terminal glutamine of CCL2. Furthermore, N-terminal Gly29, Ala30, Cys32, and His33 of CCR2, closed to the C₂Mab-9 epitope, are also involved in the interaction with CCL2. ²⁴ Therefore, there is a possibility that C₂Mab-9 affects the CCL2–CCR2 interaction. The ₂₉-GAPCH-₃₃ sequence in human CCR2 is conserved as ₄₂-GEPCH-₄₆ in mCCR2. Since the C₂Mab-6 epitope is apart from the CCL2-binding sequence in mouse, further studies are essential to evaluate the biological activity of C₂Mab-6.

The inhibition of CCR2 functions has been reported to enhance the antitumor immunity of immune checkpoint blockades including anti-programmed cell death-1 mAb in mouse model. ¹¹ C₂Mab-6 could be useful for the investigation of mCCR2 expression in immune cells, and the combination therapy with immune checkpoint blockade in mice tumor models.