Epitope Mapping of the Anti-Human CCR2 Monoclonal Antibody C 2 Mab-9

Introduction

Chemokines play pivotal roles in cellular function, including immune responses.^(1,2) They are divided into four different subfamilies of XC, CC, CXC, and CX3C, depending on the number and position of cysteine residues at the N-terminus.^(3,4) Chemokine receptors are G protein-coupled receptor (GPCR) with seven transmembrane regions.

CC chemokine receptor type-2 (CCR2) is expressed in multiple cells, including epithelial cells, monocytes, macrophages, and dendritic cells. CCR2 is involved in the regulation of migration and infiltration of immune-related cells.^(5–8) CCR2 is the primary receptor of C-C motif chemokine 2 (CCL2)/monocyte chemoattractant protein-1. CCL2 also plays an important role in attracting the key immune regulators, including T lymphocytes, natural killer cells, and monocytes.^(5,9) CCL2-CCR2 axis is correlated with many diseases such as immune disorders and cancers.^(10,11)

In respiratory organs, CCL2 and CCR2-expressing macrophages and neutrophils contribute to the innate immune responses during viral and bacterial infection.⁽¹²⁾ CCR2 induces neutrophil organ entry in sepsis.⁽¹³⁾ High CCR2 levels in blood samples have been detected in patients with severe COVID-19.⁽¹⁴⁾ Furthermore, CCL2 has been reported to be increased in several tumors, such as bladder,⁽¹⁵⁾ bone,⁽¹⁶⁾ and inflammatory breast cancers.⁽¹⁷⁾ High CCR2 expression has been confirmed in the invasive lesion of breast cancers as well as CCL2 expression.⁽¹⁸⁾ Inhibition of CCR2 has been reported to enhance the effectiveness of immune checkpoint inhibitors, such as an anti-programmed-cell death-1 monoclonal antibody (mAb) in mouse tumor models, and clinical trials are ongoing.^(15,19)

We have produced numerous mAbs against membrane proteins, including EGFR,⁽²⁰⁾ HER2,⁽²¹⁾ HER3,⁽²²⁾ CD20,⁽²³⁾ CD44,⁽²⁴⁾ CD133,⁽²⁵⁾ and podoplanin,^(26–30) and determined their epitopes.^(31–34) Moreover, we have also developed anti-GPCR mAbs, including anti-mouse CCR2,⁽³⁵⁾ mouse CCR3,⁽³⁶⁾ mouse CCR4,⁽³⁷⁾ mouse CCR8,⁽³⁸⁾ and human CCR9,⁽³⁹⁾ and also determined the binding epitope.⁽⁴⁰⁾ In this study, we performed the epitope mapping of C₂Mab-9 by using enzyme-linked immunosorbent assay (ELISA) and flow cytometric methods.

Materials and Methods

Results

Epitope mapping using N-terminal hCCR2 peptides

In our previous study, we developed an anti-hCCR2 mAb (clone C₂Mab-9) using synthetic peptide immunization method.⁽⁴¹⁾ C₂Mab-9 is suitable for in vitro applications, including flow cytometry and immunocytochemistry. To characterize the binding epitope of C₂Mab-9 for hCCR2, we synthesized three N-terminal peptides: 1–19 amino acids (aa), 11–29 aa, and 21–39 aa (Table 1). The results of ELISA demonstrated that C₂Mab-9 reacted with 11–29 aa and 21–39 aa of hCCR2 (Fig. 1A). These results are summarized in Figure 1B.

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FIG. 1.

Determination of the C₂Mab-9 epitope for hCCR2 by ELISA using N-terminal peptides. (A) N-terminal synthesized peptides of hCCR2 were immobilized on immunoplates. The plates were incubated with C₂Mab-9 (1 μg/mL) followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins. (B) Schematic illustration of hCCR2 and the C₂Mab-9 epitope. ELISA, enzyme-linked immunosorbent assay; hCCR2, human CC chemokine receptor 2.

Epitope mapping using alanine-substituted hCCR2 peptides

We further synthesized 20 different alanine-substituted hCCR2 peptides (Table 2). The results of ELISA demonstrated that C₂Mab-9 reacted with point mutants, such as T21A, T22A, Y28A, G29A, A30G, P31A, S32A, H33A, K34A, F35A, D36A, V37A, K38A, Q39A, and I40A, as well as the 21–40 aa wild-type sequence (positive control) (Fig. 2A). In contrast, C₂Mab-9 did not bind to point mutants, such as the F23A, F24A, D25A, Y26A, and D27A (Fig. 2A), indicating that Phe23, Phe24, Asp25, Tyr26, and Asp27 were determined to be the critical aa, which are included in the C₂Mab-9 epitope. The results are summarized in Figure 2B.

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FIG. 2.

Determination of the C₂Mab-9 epitope for hCCR2 by ELISA using point mutants. (A) Synthesized peptides of hCCR2 were immobilized on immunoplates. The plates were incubated with C₂Mab-9 (1 μg/mL), followed by peroxidase-conjugated anti-mouse immunoglobulins. (B) Schematic illustration of hCCR2 and the C₂Mab-9 epitope. The C₂Mab-9 epitope of hCCR2 involves Phe23, Phe24, Asp25, Tyr26, and Asp27.

Flow cytometry using C₂Mab-9 and alanine-substituted hCCR2 peptides

We then performed a blocking assay using flow cytometry. C₂Mab-9 reacted with the U937 cell line (Fig. 3A). This reaction was completely neutralized by T22A and D27A. In contrast, F23A, F24A, D25A, and Y26A did not block the reaction of C₂Mab-9 for U937 cells (Fig. 3A), indicating that Phe23, Phe24, Asp25, and Tyr26 of hCCR2 are critical for C₂Mab-9 detection. The results are summarized in Figure 3B.

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FIG. 3.

Flow cytometry using C₂Mab-9 and peptides of hCCR2. (A) C₂Mab-9 (1 μg/mL), C₂Mab-9 (1 μg/mL) plus peptides (T22A, F23A, F24A, D25A, Y26A, and D27A; 10 μg/mL), or control (blocking buffer) were reacted with U937 cells for 30 min at 4°C, followed by the addition of secondary antibodies. (B) Schematic illustration of hCCR2 and the C₂Mab-9 epitope. The C₂Mab-9 epitope of hCCR2 involves Phe23, Phe24, Asp25, and Tyr26.

Discussion

Design of immunogen and epitope identification are essential for the development of mAbs and vaccines.^(42,43) Furthermore, the determination of the mAb epitope can lead to the development of peptide tag systems for protein extraction, purification, or detection assays.^(44–47)

We previously developed an anti-human CCR9 mAb (clone C₉Mab-1) by using Cell-Based Immunization and Screening method and clarified that the epitope of C₉Mab-1 is located on the N-terminus of the human CCR9.^(39,40) Then, we have developed several anti-GPCR mAbs, including anti-mouse CCR2,⁽³⁵⁾ human CCR2,⁽⁴¹⁾ mouse CCR3,⁽⁴⁸⁾ and mouse CCR4⁽³⁷⁾ by immunizing mice or rats with the N-terminal peptides of GPCRs.

In this study, we identified the critical epitope of C₂Mab-9 as Phe23, Phe24, Asp25, Tyr26, and Asp27 using ELISA (Fig. 2). In contrast, blocking assay using flow cytometry revealed that Phe23, Phe24, Asp25, and Tyr26 of hCCR2 were critical epitope of C₂Mab-9 (Fig. 3). This discrepancy might be caused by the condition of peptides: solid phase of ELISA or liquid phase of flow cytometry. We have previously developed various novel epitope mapping system, named RIEDL insertion for epitope mapping (REMAP)^(49–52) and histidine-tag insertion for epitope mapping (HisMAP) method.⁽⁵³⁾ These methods could be applied to determine linear and structural epitopes. Therefore, we should determine the binding epitope of C₂Mab-9 using REMAP and HisMAP methods in the future study.

CCR2 is a seven-transmembrane receptor with four extracellular regions (Figs. 2B and 3B). N-terminal domains of some GPCRs, such as CCR2, CCR3, CCR5, and CXCR1, have been determined as their ligand-binding sites.⁽⁵⁴⁾ The several aa in the N-terminal region of CCR2B (predominant isoform of CCR2) were reported to be important for CCL2-triggered cell migration and lamellipodium formation,⁽⁵⁵⁾ indicating that C₂Mab-9 might be advantageous for the functional study about the CCL2-CCR2 axis.