Epitope Mapping of Anti-Mouse C-C Motif Chemokine Receptor 1 Monoclonal Antibodies Developed by the Cell-Based Immunization and Screening Method

Abstract

The C-C motif chemokine receptor 1 (CCR1) plays key roles in guiding leukocyte movement during immune surveillance and inflammatory responses. Targeting CCR1 is a promising approach for treating autoimmune diseases and cancers. We previously developed monoclonal antibodies against mouse CCR1 (mCCR1) (clones C₁Mab-2 and C₁Mab-6) for use in flow cytometry and Western blotting. However, the specific binding sites have not yet been identified. This study examined the binding epitope of C₁Mab-2 and C₁Mab-6 using flow cytometry. Analysis of mCCR1 mutants with altered extracellular domains showed that C₁Mab-2 and C₁Mab-6 bind to the N-terminal region of mCCR1. Additionally, PA-tag substitution experiments identified the epitope as 1st Met and amino acids 2–13 of mCCR1. Further alanine (or glycine) scanning within the N-terminal region (amino acids 2–13) demonstrated that Glu2, Asp5, and Phe6 are essential for recognition by C₁Mab-2, and Glu2, Ile3, and Asp5 are crucial for recognition by C₁Mab-6 in flow cytometry and Western blotting. These findings contribute to the understanding of mCCR1 and C₁Mab interaction.

Keywords

mouse CCR1 monoclonal antibody epitope mapping alanine scanning flow cytometry

Introduction

Chemokine receptors are part of the seven-transmembrane G protein–coupled receptor family and play essential roles in controlling leukocyte trafficking during immune surveillance and inflammation responses.¹ Chemokines are classified into the CC, CXC, XC, and CX3C subfamilies based on the arrangement of the first two conserved cysteine residues. CC chemokines (CCL1–CCL28) are identified by C-C chemokine receptors CCR1–CCR10.² Upon ligand engagement, chemokine receptors typically activate heterotrimeric G protein signaling pathways and recruit β-arrestins for receptor regulation endocytosis.^3,4

CCR1 is a crucial mediator of inflammatory responses and plays a role in the development of autoimmune diseases.^1,5 Accordingly, CCR1 is considered a promising therapeutic target for allergic and autoimmune disorders.⁶ Notably, CCR1 shows ligand promiscuity, allowing it to recognize multiple CC chemokines, including CCL3, CCL5, CCL7, CCL8, CCL13–16, and CCL23.^2,7,8

Structural elucidation of chemokine receptor activation is essential for rational drug design targeting the chemokine system. Among CCR family members, both inactive and ligand-bound active conformations have been resolved for CCR2 and CCR5.^9–12 Additionally, ligand-bound active-state structures of CCR6 and CCR8, along with inactive-state structures of CCR7 and CCR9, have been reported.^13–16 The structure of the CCL15–CCR1 complex showed that extracellular loops (ECL2 and ECL3) contain key factors for ligand recognition, setting CCR1 apart from other chemokine receptor–ligand interactions and offering new insights into chemokine-binding mechanisms.⁹ Furthermore, structural analyses of CCR8 in complex with either its endogenous ligand CCL1 or an antagonistic monoclonal antibody (mAb) have elucidated mechanisms underlying receptor activation and antibody-mediated inhibition.¹³ These findings highlight the usefulness of antichemokine receptor mAbs with specific epitopes for structural and functional studies.

The Cell-Based Immunization and Screening (CBIS) method is a strategy for generating a variety of mAbs against membrane proteins. In this approach, antigen-overexpressed cells are immunized, and the hybridoma supernatants are then screened using flow cytometry-based high-throughput techniques. We have developed several mAbs targeting chemokine receptors, such as mouse CCR1 (mCCR1) and mouse CCR5 (mCCR5), utilizing the CBIS method.^17,18 These mAbs are expected to recognize various epitopes, including linear and conformational ones. However, their specific binding epitopes are still undefined.

We previously reported the flow cytometry-based epitope mapping of an anti-mCCR1 mAb (S15040E) and an anti-mCCR8 mAb (C₈Mab-2) using mutants with the extracellular domain substituted and alanine scanning.^19,20 Using the epitope identification strategy, we aimed to determine the binding epitopes of anti-mCCR1 mAbs (C₁Mab-2 and C₁Mab-6) through flow cytometry and Western blotting.

Materials and Methods

Plasmid construction

pCAG-Ble-mCCR1 and pCAG-Ble-mouse CCR5 (mCCR5) were generated as previously described.^17,18 Chimeric mutants, including mCCR5 (mCCR1p2–34), mCCR5 (mCCR1p92–107), mCCR5 (mCCR1p172–197), and mCCR5 (mCCR1p265–281), were produced as described previously.¹⁹ Alanine (or glycine)-substituted mutants of mCCR1 were generated using QuikChange Lightning Site-Directed Mutagenesis Kits (Agilent Technologies Inc., Santa Clara, CA, USA). PA-tag (GVAMPGAEDDVV)-substituted mCCR1 mutants were created using a PCR-based method. The PCR fragments containing the desired mutations were inserted into the pCAG-Ble or pCAG-neo vectors (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

Cell line and plasmid transfection

The chimeric, PA-tag-substituted, and point-mutant plasmids were transfected into Chinese hamster ovary (CHO)-K1 cells (American Type Culture Collection, Manassas, VA, USA) using the Neon Transfection System (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Antibodies

C₁Mab-2 and C₁Mab-6 were established as described previously.¹⁸ C₅Mab-2 and C₅Mab-4 were also developed by our laboratory.^17,21 An anti-mCCR1 mAb (clone S15040E) was purchased from BioLegend (San Diego, CA, USA).

Flow cytometry

Cells were collected after brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin in phosphate-buffered saline, 2 × 10⁵ cells were treated with 1 µg/mL of C₁Mab-2, C₁Mab-6, C₅Mab-2, C₅Mab-4, or S15040E for 30 minutes at 4°C, followed by incubation with Alexa Fluor 488-conjugated anti-rat IgG (1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA). Fluorescence data (from a total of 5000 cells per sample) were collected using the SA3800 Cell Analyzer (Sony Corp., Tokyo, Japan). Using FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA), single cells were gated based on side scatter versus forward scatter, and the fluorescence intensity was plotted.

Western blotting

Cell lysates were boiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). Proteins (10 µg/lane) were electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking with 4% nonfat milk (Nacalai Tesque, Inc., Kyoto, Japan), PVDF membranes were incubated with 1 µg/mL of C₁Mab-2, C₁Mab-6, or an anti-IDH1 mAb (clone RcMab-1), followed by incubation with horseradish peroxidase-conjugated anti-rat IgG (1:10,000; Sigma-Aldrich Corp., St. Louis, MO). Finally, protein bands were detected with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

Results

Determination of the epitopes of anti-mCCR1 mAbs (C₁Mab-2 and C₁Mab-6) by flow cytometry using chimeric proteins

Anti-mCCR1 mAbs, clone C₁Mab-2 (rat IgG_2b, lambda) and C₁Mab-6 (rat IgG_2b, kappa)¹⁸ were previously established and are applicable for flow cytometry and Western blotting (http://www.med-tohoku-antibody.com/topics/001_paper_antibody_PDIS.htm). To investigate the binding epitopes of C₁Mab-2 and C₁Mab-6, we focused on four extracellular regions of mCCR1: the N-terminal region [amino acid (aa) 1–34], ECL1 (aa 92–107), ECL2 (aa 172–197), and ECL3 (aa 265–281). These regions of mCCR1 were replaced with the corresponding regions of mCCR5, which is highly homologous to mCCR1. As shown in Figure 1, plasmids encoding mCCR5 with these specific mCCR1 segments—mCCR1p1–34, mCCR1p92–107, mCCR1p172–197, and mCCR1p265–281—were created. The chimeric proteins were transiently expressed in CHO-K1 cells, and their reactivities to C₁Mab-2 and C₁Mab-6 were analyzed via flow cytometry (Fig. 2A). Both C₁Mab-2 and C₁Mab-6 reacted with mCCR5 containing mCCR1p1–34 but not with those containing mCCR1p92–107, mCCR1p172–197, or mCCR1p265–281 (Fig. 2A and B). The cell-surface expression of each mutant was confirmed using anti-mCCR5 mAbs, C₅Mab-2, and C₅Mab-4, which recognize the mCCR5 ECL2 and N-terminal regions, respectively (Fig. 2C and D). These results suggest that C₁Mab-2 and C₁Mab-6 recognize the N-terminal region of mCCR1.

FIG. 1.

Schematic illustration of chimeric proteins. The four extracellular regions of mCCR1, including the N-terminal region (aa 1–34), ECL1 (aa 92–107), ECL2 (aa 172–197), and ECL3 (aa 265–281), were substituted into the corresponding regions of mCCR5. aa, amino acid; ECL, extracellular loop.

FIG. 2.

Determination of the epitope of anti-mCCR1 mAbs, C₁Mab-2, and C₁Mab-6 by flow cytometry using chimeric proteins. CHO-K1 cells transiently expressing the chimeric proteins were treated with C₁Mab-2 (1 µg/mL, A), C₁Mab-6 (1 µg/mL, B), C₅Mab-2 (1 µg/mL, C), C₅Mab-4 (1 µg/mL, D), or blocking buffer for 30 minutes at 4°C, followed by the addition of Alexa Fluor 488-conjugated anti-rat IgG. Red lines indicate cells treated with C₁Mab-2, C₁Mab-6, C₅Mab-2, or C₅Mab-4, while black lines indicate cells treated with blocking buffer as a negative control. The normalized reactivity (geometric mean of C₁Mab-2 or C₁Mab-6/geometric mean of C₅Mab-2 or C₅Mab-4) is indicated in each histogram (A and B). Experiments were conducted twice. Representative histograms are shown.

Determination of the C₁Mab-2 and C₁Mab-6 epitopes by flow cytometry using PA-tag substitution

Next, we constructed PA-tag-substituted mCCR1 in the N-terminal region. As shown in Figure 3, PA-tag-substituted mCCR1 mutants on amino acids 2–13 (mCCR1-2PA13), 12–23 (mCCR1-12PA23), and 23–34 (mCCR1-23PA34) were created. Additionally, the 1st Met-substituted mCCR1 by PA-tag [PA-mCCR1 (2–355)] was produced. The PA-tag-substituted mCCR1 mutants were transiently expressed in CHO-K1 cells, and their reactivity to C₁Mab-2 and C₁Mab-6 was analyzed using flow cytometry. As shown in Figure 4, neither C₁Mab-2 nor C₁Mab-6 reacted with mCCR1-2PA13 nor PA-mCCR1 (2–355), but both reacted with mCCR1-12PA23 and mCCR1-23PA34. These results suggest that the epitopes of C₁Mab-2 nor C₁Mab-6 are located in the 1st Met and amino acids 2–13 of mCCR1.

FIG. 3.

The illustration of PA-tag-substituted mutants of mCCR1.

FIG. 4.

Determination of the C₁Mab-2 and C₁Mab-6 epitopes by flow cytometry using PA-tag-substituted mutants of mCCR1. CHO-K1 cells transiently expressing PA-tag-substituted mCCR1 mutants were treated with C₁Mab-2 (1 µg/mL, A), C₁Mab-6 (1 µg/mL, B), S15040E (1 µg/mL, C), or blocking buffer for 30 minutes at 4°C. Subsequently, the cells were incubated with Alexa Fluor 488-conjugated anti-rat IgG. Red lines indicate cells treated with C₁Mab-2, C₁Mab-6, or S15040E, while black lines represent cells treated with blocking buffer as a negative control. The normalized reactivity (geometric mean of C₁Mab-2 or C₁Mab-6/geometric mean of S15040E) is indicated in each histogram (A and B). Experiments were conducted twice. Representative histograms are shown.

Determination of the C₁Mab-2 and C₁Mab-6 epitopes by flow cytometry using alanine scanning

Next, alanine scanning was performed on the N-terminal region (aa 2–13) of mCCR1. Twelve mutants with alanine (or glycine) substitutions in mCCR1 were created (Fig. 5), and these mutant proteins were transiently expressed in CHO-K1 cells. Reactivity with C₁Mab-2 and C₁Mab-6 was assessed by flow cytometry. As shown in Figure 6A, C₁Mab-2 did not react with two mutants (E2A and F6A), and the reactivity of C₁Mab-2 to the D5A mutant was reduced. Similarly, C₁Mab-6 did not react with two mutants (E2A and D5A), and the reactivity of C₁Mab-6 to the I3A mutant was reduced. (Fig. 6B). The cell surface expression of each mutant was confirmed with S15040E, which recognizes ECL2 (Fig. 6C).¹⁹ The normalized reactivity of C₁Mab-2 and C₁Mab-6 (Fig. 6D) confirmed that Glu2, Asp5, and Phe6 are essential for recognition by C₁Mab-2, and Glu2, Ile3, and Asp5 are crucial for recognition by C₁Mab-6 in flow cytometry.

FIG. 5.

The illustration of alanine (or glycine)-substituted mutants of mCCR1.

FIG. 6.

Determination of C₁Mab-2 and C₁Mab-6 epitopes by flow cytometry using alanine scanning. CHO-K1 transiently expressed mCCR1 mutants and wild-type (WT) were treated with C₁Mab-2 (1 µg/mL, A), C₁Mab-6 (1 µg/mL, B), S15040E (1 µg/mL, C), or blocking buffer for 30 minutes at 4°C. Then, the cells were treated with Alexa Fluor 488-conjugated anti-ratIgG. Red lines indicate the cells treated with C₁Mab-2, C₁Mab-6, or S15040E, while black lines show the cells treated with blocking buffer as the negative control. Experiments were conducted three times. Representative histograms are shown. (D) The normalized reactivity (geometric mean of C₁Mab-2 or C₁Mab-6/geometric mean of S15040E) was calculated.

Determination of the C₁Mab-2 and C₁Mab-6 epitopes by Western blotting using alanine scanning

Since C₁Mab-2 and C₁Mab-6 are suitable for Western blotting, the lysates of mutants were also examined using Western blotting. As shown in Figure 7A, C₁Mab-2 detected 45–48 kDa and slower-migrating smear bands, which completely disappeared in the lysates of three mutants (E2A, D5A, and F6A). Furthermore, C₁Mab-6 detected 45–48 kDa and slower migrating smear bands, which completely disappeared in the lysates of three mutants (E2A, I3A, and D5A; Fig. 7B). An anti-IDH1mAb, RcMab-1, was used as an internal control (Fig. 7C). These results were consistent with that of flow cytometry.

FIG. 7.

Determination of C₁Mab-2 and C₁Mab-6 epitopes by Western blotting. Cell lysates (10 μg/lane) from CHO-K1 cells transiently expressing mCCR1 mutants and wild-type (WT) were electrophoresed and transferred onto polyvinylidene difluoride membranes. The membranes were incubated with C₁Mab-2 (1 μg/mL, A), C₁Mab-6 (1 μg/mL, B), or RcMab-1 (an anti-IDH1 mAb, 1 μg/mL, C), followed by treatment with anti-rat IgG conjugated to horseradish peroxidase. Experiments were conducted three times. Representative images are shown.

Discussion

This study conducted flow cytometry-based epitope mapping of anti-mCCR1 mAbs (C₁Mab-2 and C₁Mab-6) using chimeric proteins (Figs. 1 and 2) and PA-tag-substituted mutants (Figs. 3 and 4). Subsequently, alanine scanning revealed that Glu2, Asp5, and Phe6 are essential for recognition by C₁Mab-2 in flow cytometry (Fig. 6A) and Western blotting (Fig. 7A). For C₁Mab-6, Glu2, Ile3, and Asp5 are vital in flow cytometry (Fig. 6B) and Western blotting (Fig. 7B). Additionally, we found that C₁Mab-2 and C₁Mab-6 did not recognize the 1st Met-substituted mCCR1 [PA-mCCR1 (2–355)] in flow cytometry (Fig. 4), indicating that the 1st Met is also essential for the epitope. Figure 8 summarizes the results and illustrates the complementarity-determining regions (CDRs) of C₁Mab-2 and C₁Mab-6. Although C₁Mab-2 and C₁Mab-6 have different CDR sequences, they recognize similar epitopes in the mCCR1 N-terminal region.

FIG. 8.

The schematic illustration of C₁Mab-2 and C₁Mab-6 epitopes. (A) Met1 is essential for the recognition by C₁Mab-2 in flow cytometry (underlined), and Glu2, Asp5, and Phe6 are essential for the recognition by C₁Mab-2 in flow cytometry (underlined) and Western blotting (yellow). The CDR sequences are indicated. (B) Met1 is essential for the recognition by C₁Mab-6 in flow cytometry (underlined), and Glu2, Ile3, and Asp5 are essential for the recognition by C₁Mab-6 in flow cytometry (underlined) and Western blotting (yellow). The CDR sequences of C₁Mab-6 are indicated. CDR, complementarity-determining region.

The N-terminal region of the chemokine receptor is known as chemokine receptor site 1 (CRS1), which mainly mediates interactions with the chemokine N-loop/β3-strands and 40-second loop. These contacts are crucial for establishing the initial binding affinity and specificity between the chemokine and its cognate receptor.²² CRS1 adopts a flexible and unstructured conformation when not in the presence of ligands.^23–28 The identification of the epitope would contribute to the understanding of mAb-epitope interaction.

Chemokine–receptor interaction is a multistep process generally described by the two-site, two-step model within CRS1 and CRS2. In this model, chemokine–CRS1 interactions are initially established and facilitate ligand binding in the absence of receptor activation. After the initial docking at CRS1, a second interaction occurs at CRS2, where the chemokine N-terminus engages the deeper orthosteric binding pocket of the receptor. This subsequent interaction is essential for receptor activation, triggering the conformational changes needed for downstream intracellular processes signaling.²² Since human CCR1 (hCCR1) N-terminal peptide (aa 1–29) serves as CRS1 to interact with the ligands,²⁹ corresponding mCCR1 N-terminus is thought to be involved in the ligand binding. Therefore, C₁Mab-2 and C₁Mab-6 are expected to neutralize the ligand binding. Further binding and functional studies are essential to prove the neutralizing effects.

CCR1 has been demonstrated as a drug target for the treatment of autoimmune diseases and tumors.⁶ Recently, blocking mCCR1 with a neutralizing anti-CCR1 mAb (clone KM5908) in protumorigenic myeloid cells demonstrated therapeutic efficacy in a mouse syngeneic model of colorectal cancer.³⁰ KM5908 was established by immunizing Ccr1 knockout mice with cells overexpressed hCCR1 and recognized both hCCR1 and mCCR1.³¹ Although KM5908 was selected based on its inhibitory activity on the chemotaxis of the THP-1 monocytic cell line toward CCL15 gradient,³¹ the binding epitope has not been clarified. Our epitope mapping system will help identify the KM5908 epitope, which is important for comparing properties with C₁Mab-2 and C₁Mab-6.

Authors’ Contributions

A.O. performed the experiments. M.K.K. and Y.K. designed the experiments. H.S. and Y.K. wrote the article. All authors have read and agreed to the published version of the article.

Footnotes

Author Disclosure Statement

The authors have no conflicts of interest.

Funding Information

This research was supported in part by the Japan Agency for Medical Research and Development (AMED) under grant numbers: JP26am0521010 (to Y.K.), JP26ama121008 (to Y.K.), JP25ama221153 (to Y.K.), JP25ama221339 (to Y.K.), and JP25bm1123027 (to Y.K.) and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant no. 25K10553 (to Y.K.).

References

1. Griffith

, Sokol

, Luster

. Chemokines and chemokine receptors: Positioning cells for host defense and immunity. Annu Rev Immunol 2014;32:659–702; doi: 10.1146/annurev-immunol-032713-120145

2. Stone

, Hayward

, Huang

, et al. Mechanisms of regulation of the chemokine-receptor network. Int J Mol Sci 2017;18(2):342; doi: 10.3390/ijms18020342

3. Weis

, Kobilka

. The molecular basis of G protein-coupled receptor activation. Annu Rev Biochem 2018;87:897–919; doi: 10.1146/annurev-biochem-060614-033910

4. Defea

. Beta-arrestins and heterotrimeric G-proteins: Collaborators and competitors in signal transduction. Br J Pharmacol 2008;153(Suppl 1):S298–S309; doi: 10.1038/sj.bjp.0707508

5. Tian

, Yan

, Guo

, et al. Inflammatory role of CCR1 in the central nervous system. Neuroimmunomodulation 2024;31(1):173–182; doi: 10.1159/000540460

6. Scholten

, Canals

, Maussang

, et al. Pharmacological modulation of chemokine receptor function. Br J Pharmacol 2012;165(6):1617–1643; doi: 10.1111/j.1476-5381.2011.01551.x

7. Schall

, Proudfoot

. Overcoming hurdles in developing successful drugs targeting chemokine receptors. Nat Rev Immunol 2011;11(5):355–363; doi: 10.1038/nri2972

8. Murphy

, Baggiolini

, Charo

, et al. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev 2000;52(1):145–176.

9. Shao

, Shen

, Yao

, et al. Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1. Nat Chem Biol 2022;18(3):264–271; doi: 10.1038/s41589-021-00918-z

10.

10. Isaikina

, Tsai

, Dietz

, et al. Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist. Sci Adv 2021;7(25):eabg8685; doi: 10.1126/sciadv.abg8685

11.

11. Zheng

, Qin

, Zacarías

, et al. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature 2016;540(7633):458–461; doi: 10.1038/nature20605

12.

12. Tan

, Zhu

, Li

, et al. Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex. Science 2013;341(6152):1387–1390; doi: 10.1126/science.1241475

13.

13. Sun

, Sun

, Janezic

, et al. Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8. Nat Commun 2023;14(1):7940; doi: 10.1038/s41467-023-43601-8

14.

14. Wasilko

, Johnson

, Ammirati

, et al. Structural basis for chemokine receptor CCR6 activation by the endogenous protein ligand CCL20. Nat Commun 2020;11(1):3031; doi: 10.1038/s41467-020-16820-6

15.

15. Jaeger

, Bruenle

, Weinert

, et al. Structural basis for allosteric ligand recognition in the human CC chemokine receptor 7. Cell 2019;178(5):1222–1230.e1210; doi: 10.1016/j.cell.2019.07.028

16.

16. Oswald

, Rappas

, Kean

, et al. Intracellular allosteric antagonism of the CCR9 receptor. Nature 2016;540(7633):462–465; doi: 10.1038/nature20606

17.

17. Suzuki

, Tanaka

, Li

, et al. Development of a sensitive anti-mouse CCR5 monoclonal antibody for flow cytometry. Monoclon Antib Immunodiagn Immunother 2024;43(4):96–100; doi: 10.1089/mab.2024.0004

18.

18. Ouchida

, Isoda

, Nakamura

, et al. Establishment of a novel anti-mouse CCR1 monoclonal antibody C(1)Mab-6. Monoclon Antib Immunodiagn Immunother 2024;43(2):67–74; doi: 10.1089/mab.2023.0032

19.

19. Okada

, Suzuki

, Arimori

, et al. Epitope analysis of an anti-mouse CCR1 monoclonal antibody S15040E using flow cytometry. Biochem Biophys Rep 2025;44:102265; doi: 10.1016/j.bbrep.2025.102265

20.

20. Kobayashi

, Suzuki

, Tanaka

, et al. Epitope mapping of an anti-mouse CCR8 monoclonal antibody C(8)Mab-2 using flow cytometry. Monoclon Antib Immunodiagn Immunother 2024;43(4):101–107; doi: 10.1089/mab.2024.0002

21.

21. Ubukata

, Suzuki

, Tanaka

, et al. Development of sensitive anti-mouse CCR5 monoclonal antibodies using the N-terminal peptide immunization. Monoclon Antib Immunodiagn Immunother 2024;43(4):112–118; doi: 10.1089/mab.2024.0009

22.

22. Kayastha

, Zhou

, Brünle

. Structural perspectives on chemokine receptors. Biochem Soc Trans 2024;52(3):1011–1024; doi: 10.1042/bst20230358

23.

23. Millard

, Ludeman

, Canals

, et al. Structural basis of receptor sulfotyrosine recognition by a CC chemokine: The N-terminal region of CCR3 bound to CCL11/eotaxin-1. Structure 2014;22(11):1571–1581; doi: 10.1016/j.str.2014.08.023

24.

24. Chaudhuri

, Basu

, Haldar

, et al. Organization and dynamics of the N-terminal domain of chemokine receptor CXCR1 in reverse micelles: Effect of graded hydration. J Phys Chem B 2013;117(5):1225–1233; doi: 10.1021/jp3095352

25.

25. Haldar

, Raghuraman

, Namani

, et al. Membrane interaction of the N-terminal domain of chemokine receptor CXCR1. Biochim Biophys Acta 2010;1798(6):1056–1061; doi: 10.1016/j.bbamem.2010.02.029

26.

26. Veldkamp

, Seibert

, Peterson

, et al. Structural basis of CXCR4 sulfotyrosine recognition by the chemokine SDF-1/CXCL12. Sci Signal 2008;1(37):ra4; doi: 10.1126/scisignal.1160755

27.

27. Fernando

, Nagle

, Rajarathnam

. Thermodynamic characterization of interleukin-8 monomer binding to CXCR1 receptor N-terminal domain. FEBS J 2007;274(1):241–251; doi: 10.1111/j.1742-4658.2006.05579.x

28.

28. Duma

, Häussinger

, Rogowski

, et al. Recognition of RANTES by extracellular parts of the CCR5 receptor. J Mol Biol 2007;365(4):1063–1075; doi: 10.1016/j.jmb.2006.10.040

29.

29. Sanchez

, E Huma

, Lane

, et al. Evaluation and extension of the two-site, two-step model for binding and activation of the chemokine receptor CCR1. J Biol Chem 2019;294(10):3464–3475; doi: 10.1074/jbc.RA118.006535

30.

30. Masui

, Kawada

, Itatani

, et al. Synergistic antitumor activity by dual blockade of CCR1 and CXCR2 expressed on myeloid cells within the tumor microenvironment. Br J Cancer 2024;131(1):63–76; doi: 10.1038/s41416-024-02710-x

31.

31. Kiyasu

, Kawada

, Hirai

, et al. Disruption of CCR1-mediated myeloid cell accumulation suppresses colorectal cancer progression in mice. Cancer Lett 2020;487:53–62; doi: 10.1016/j.canlet.2020.05.028

Epitope Mapping of Anti-Mouse C-C Motif Chemokine Receptor 1 Monoclonal Antibodies Developed by the Cell-Based Immunization and Screening Method

Abstract

Keywords

Introduction

Materials and Methods

Plasmid construction

Cell line and plasmid transfection

Antibodies

Flow cytometry

Western blotting

Results

Determination of the epitopes of anti-mCCR1 mAbs (C1Mab-2 and C1Mab-6) by flow cytometry using chimeric proteins

Determination of the C1Mab-2 and C1Mab-6 epitopes by flow cytometry using PA-tag substitution

Determination of the C1Mab-2 and C1Mab-6 epitopes by flow cytometry using alanine scanning

Determination of the C1Mab-2 and C1Mab-6 epitopes by Western blotting using alanine scanning

Discussion

Authors’ Contributions

Footnotes

Author Disclosure Statement

Funding Information

References

Determination of the epitopes of anti-mCCR1 mAbs (C₁Mab-2 and C₁Mab-6) by flow cytometry using chimeric proteins

Determination of the C₁Mab-2 and C₁Mab-6 epitopes by flow cytometry using PA-tag substitution

Determination of the C₁Mab-2 and C₁Mab-6 epitopes by flow cytometry using alanine scanning

Determination of the C₁Mab-2 and C₁Mab-6 epitopes by Western blotting using alanine scanning