Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry

Tateyama, Nami; Asano, Teizo; Suzuki, Hiroyuki; Li, Guanjie; Yoshikawa, Takeo; Tanaka, Tomohiro; Kaneko, Mika K.; Kato, Yukinari

doi:10.3390/antib11040075

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Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry

by

Nami Tateyama

^1,†,

Teizo Asano

^1,†

,

Hiroyuki Suzuki

^2,*

,

Guanjie Li

²,

Takeo Yoshikawa

³

,

Tomohiro Tanaka

¹,

Mika K. Kaneko

¹ and

Yukinari Kato

^1,2,3,*

¹

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan

²

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan

³

Department of Pharmacology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan

^*

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Antibodies 2022, 11(4), 75; https://doi.org/10.3390/antib11040075

Submission received: 2 November 2022 / Revised: 25 November 2022 / Accepted: 29 November 2022 / Published: 2 December 2022

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Abstract

The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C₃Mab-3 (rat IgG_2a, kappa), and C₃Mab-4 (rat IgG_2a, kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C₃Mab-3, C₃Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C₃Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C₃Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C₃Mab-3, C₃Mab-4, and J073E5.

Keywords: mouse CCR3; monoclonal antibody; epitope mapping; alanine scanning; flow cytometry

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MDPI and ACS Style

Tateyama, N.; Asano, T.; Suzuki, H.; Li, G.; Yoshikawa, T.; Tanaka, T.; Kaneko, M.K.; Kato, Y. Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry. Antibodies 2022, 11, 75. https://doi.org/10.3390/antib11040075

AMA Style

Tateyama N, Asano T, Suzuki H, Li G, Yoshikawa T, Tanaka T, Kaneko MK, Kato Y. Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry. Antibodies. 2022; 11(4):75. https://doi.org/10.3390/antib11040075

Chicago/Turabian Style

Tateyama, Nami, Teizo Asano, Hiroyuki Suzuki, Guanjie Li, Takeo Yoshikawa, Tomohiro Tanaka, Mika K. Kaneko, and Yukinari Kato. 2022. "Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry" Antibodies 11, no. 4: 75. https://doi.org/10.3390/antib11040075

APA Style

Tateyama, N., Asano, T., Suzuki, H., Li, G., Yoshikawa, T., Tanaka, T., Kaneko, M. K., & Kato, Y. (2022). Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry. Antibodies, 11(4), 75. https://doi.org/10.3390/antib11040075

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Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry

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