AccScience Publishing / MI / Online First / DOI: 10.36922/mi.5664
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Establishment of a high-affinity anti-mouse CXCR5 monoclonal antibody for flow cytometry

Kenichiro Ishikawa1 ,  Hiroyuki Suzuki1 ,  Tomohiro Tanaka1 ,  Mika K. Kaneko1 ,  Yukinari Kato1*
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1 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, Japan
MI, 5664 https://doi.org/10.36922/mi.5664
Submitted: 29 October 2024 | Revised: 1 December 2024 | Accepted: 10 December 2024 | Published: 26 December 2024
© 2024 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

The CXC chemokine receptor 5 (CXCR5) is a member of the G protein-coupled receptor family that is highly expressed in B cells and a subset of T cells, such as T follicular helper cells. Various types of cancers, including non-small cell lung cancer, breast cancer, and prostate cancer, also express CXCR5. Therefore, antibodies that specifically bind to CXCR5 could be useful for clarification of the mechanisms of cancer progression. In this study, we aimed to develop high-affinity monoclonal antibodies targeting mouse CXCR5 (mCXCR5) for flow cytometry. The established anti-mCXCR5 mAb (Cx5Mab-3; rat IgG2b, kappa), demonstrated reactivity with mCXCR5-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/mCXCR5) in flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KD) of Cx5Mab-3 for CHO/mCXCR5 cell is 7.2 × 10−10 M. Furthermore, Cx5Mab-3 did not cross-react with other mouse CC, CXC, CX3C, and XC chemokine receptors. These results indicate that Cx5Mab-3 is useful for detecting mCXCR5 in flow cytometry with high affinity and specificity.

Keywords
Mouse CXC chemokine receptor 5
Monoclonal antibody
Peptide immunization
Flow cytometry
Funding
This research was supported in part by Japan Agency for Medical Research and Development (AMED) under Grant Numbers: JP24am0521010 (to Y.K.), JP24ama121008 (to Y.K.), JP24ama221339 (to Y.K.), JP23am0401013 (to Y.K.), JP24bm1123027 (to Y.K.), and JP24ck0106730 (to Y.K.), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant nos. 22K06995 (to H.S.) and 22K07224 (to Y.K).
Conflict of interest
The authors declare that they have no competing interests.
References
  1. Nelson PJ, Krensky AM. Chemokines, chemokine receptors, and allograft rejection. Immunity. 2001;14(4):377-386. doi: 10.1016/s1074-7613(01)00118-2

 

  1. Lukacs-Kornek V, Engel D, Tacke F, Kurts C. The role of chemokines and their receptors in dendritic cell biology. Front Biosci. 2008;13:2238-2252. doi: 10.2741/2838

 

  1. 3. Bonecchi R, Galliera E, Borroni EM, et al. Chemokines and chemokine receptors: An overview. Front Biosci (Landmark Ed). 2009;14(2):540-551. doi: 10.2741/3261

 

  1. Comerford I, McColl SR. Mini-review series: Focus on chemokines. Immunol Cell Biol. 2011;89(2):183-184. doi: 10.1038/icb.2010.164

 

  1. Salanga CL, Handel TM. Chemokine oligomerization and interactions with receptors and glycosaminoglycans: The role of structural dynamics in function. Exp Cell Res. 2011;317(5):590-601. doi: 10.1016/j.yexcr.2011.01.004

 

  1. Zlotnik A, Yoshie O. The chemokine superfamily revisited. Immunity. 2012;36(5):705-716. doi: 10.1016/j.immuni.2012.05.008

 

  1. Marchese A. Endocytic trafficking of chemokine receptors. Curr Opin Cell Biol. 2014;27:72-77. doi: 10.1016/j.ceb.2013.11.011

 

  1. Hughes CE, Nibbs RJB. A guide to chemokines and their receptors. Febs J. 2018;285(16):2944-2971. doi: 10.1111/febs.14466

 

  1. Ortiz Zacarías NV, Lenselink EB, IJzerman AP, Handel TM, Heitman LH. Intracellular Receptor modulation: Novel approach to target GPCRs. Trends Pharmacol Sci. 2018;39(6):547-559. doi: 10.1016/j.tips.2018.03.002

 

  1. Aragay AM, Ruiz-Gómez A, Penela P, et al. G protein-coupled receptor kinase 2 (GRK2): Mechanisms of regulation and physiological functions. FEBS Lett. 1998;430(1-2):37-40. doi: 10.1016/s0014-5793(98)00495-5

 

  1. Katritch V, Cherezov V, Stevens RC. Diversity and modularity of G protein-coupled receptor structures. Trends Pharmacol Sci. 2012;33(1):17-27. doi: 10.1016/j.tips.2011.09.003

 

  1. Krumm BE, Grisshammer R. Peptide ligand recognition by G protein-coupled receptors. Front Pharmacol. 2015;6:48. doi: 10.3389/fphar.2015.00048

 

  1. Kufareva I, Salanga CL, Handel TM. Chemokine and chemokine receptor structure and interactions: Implications for therapeutic strategies. Immunol Cell Biol. 2015;93(4):372-383. doi: 10.1038/icb.2015.15

 

  1. Proudfoot AE, Uguccioni M. Modulation of chemokine responses: Synergy and cooperativity. Front Immunol. 2016;7:183. doi: 10.3389/fimmu.2016.00183

 

  1. Miller MC, Mayo KH. Chemokines from a structural perspective. Int J Mol Sci. 2017;18(10):2088. doi: 10.3390/ijms18102088

 

  1. Gustavsson M. New insights into the structure and function of chemokine receptor: chemokine complexes from an experimental perspective. J Leukoc Biol. 2020;107(6):1115-1122. doi: 10.1002/jlb.2mr1219-288r

 

  1. Laganà M, Schlecht-Louf G, Bachelerie F. The G Protein-coupled receptor kinases (GRKs) in chemokine receptor-mediated immune cell migration: From molecular cues to physiopathology. Cells. 2021;10(1):75. doi: 10.3390/cells10010075

 

  1. Shpakov AO. Allosteric regulation of G-protein-coupled receptors: From diversity of molecular mechanisms to multiple allosteric sites and their ligands. Int J Mol Sci. 2023;24(7):6187. doi: 10.3390/ijms24076187

 

  1. Thompson MD, Reiner-Link D, Berghella A, et al. G protein-coupled receptor (GPCR) pharmacogenomics. Crit Rev Clin Lab Sci. 2024;61(8):641-684. doi: 10.1080/10408363.2024.2358304

 

  1. Conroy DM, Humbles AA, Rankin SM, et al. The role of the eosinophil-selective chemokine, eotaxin, in allergic and non-allergic airways inflammation. Mem Inst Oswaldo Cruz. 1997;92(Suppl 2):183-191. doi: 10.1590/s0074-02761997000800024

 

  1. Ward SG, Westwick J. Chemokines: Understanding their role in T-lymphocyte biology. Biochem J. 1998;333(Pt 3):457-470. doi: 10.1042/bj3330457

 

  1. Traynor TR, Huffnagle GB. Role of chemokines in fungal infections. Med Mycol. 2001;39(1):41-50. doi: 10.1080/mmy.39.1.41.50

 

  1. Chensue SW. Molecular machinations: Chemokine signals in host-pathogen interactions. Clin Microbiol Rev. 2001;14(4):821-835. doi: 10.1128/cmr.14.4.821-835.2001

 

  1. Ransohoff RM. Chemokines and chemokine receptors: Standing at the crossroads of immunobiology and neurobiology. Immunity. 2009;31(5):711-721. doi: 10.1016/j.immuni.2009.09.010

 

  1. Wang X, Sharp JS, Handel TM, Prestegard JH. Chemokine oligomerization in cell signaling and migration. Prog Mol Biol Transl Sci. 2013;117:531-578. doi: 10.1016/b978-0-12-386931-9.00020-9

 

  1. Sokol CL, Luster AD. The chemokine system in innate immunity. Cold Spring Harb Perspect Biol. 2015;7(5):a016303. doi: 10.1101/cshperspect.a016303

 

  1. Luther SA, Cyster JG. Chemokines as regulators of T cell differentiation. Nat Immunol. 2001;2(2):102-107. doi: 10.1038/84205

 

  1. Allegretti M, Cesta MC, Locati M. Allosteric modulation of chemoattractant receptors. Front Immunol. 2016;7:170. doi: 10.3389/fimmu.2016.00170

 

  1. Schulz O, Hammerschmidt SI, Moschovakis GL, Förster R. Chemokines and chemokine receptors in lymphoid tissue dynamics. Annu Rev Immunol. 2016;34:203-242. doi: 10.1146/annurev-immunol-041015-055649

 

  1. Stone MJ, Hayward JA, Huang C, Huma ZE, Sanchez J. Mechanisms of regulation of the chemokine-receptor network. Int J Mol Sci. 2017;18(2):342. doi: 10.3390/ijms18020342

 

  1. López-Cotarelo P, Gómez-Moreira C, Criado- García O, Sánchez L, Rodríguez-Fernández JL. Beyond chemoattraction: Multifunctionality of chemokine receptors in leukocytes. Trends Immunol. 2017;38(12):927-941. doi: 10.1016/j.it.2017.08.004

 

  1. Laufer JM, Legler DF. Beyond migration-Chemokines in lymphocyte priming, differentiation, and modulating effector functions. J Leukoc Biol. 2018;104(2):301-312. doi: 10.1002/jlb.2mr1217-494r

 

  1. Cecchinato V, Uguccioni M. Insight on the regulation of chemokine activities. J Leukoc Biol. 2018;104(2):295-300. doi: 10.1002/jlb.3mr0118-014r

 

  1. Rajarathnam K, Schnoor M, Richardson RM, Rajagopal S. How do chemokines navigate neutrophils to the target site: Dissecting the structural mechanisms and signaling pathways. Cell Signal. 2019;54:69-80. doi: 10.1016/j.cellsig.2018.11.004

 

  1. Groom JR. Regulators of T-cell fate: Integration of cell migration, differentiation and function. Immunol Rev. 2019;289(1):101-114. doi: 10.1111/imr.12742

 

  1. Graham GJ, Handel TM, Proudfoot AEI. Leukocyte adhesion: Reconceptualizing chemokine presentation by glycosaminoglycans. Trends Immunol. 2019;40(6):472-481. doi: 10.1016/j.it.2019.03.009

 

  1. Michael M, Vermeren S. A neutrophil-centric view of chemotaxis. Essays Biochem. 2019;63(5):607-618. doi: 10.1042/ebc20190011

 

  1. Crijns H, Vanheule V, Proost P. Targeting chemokine-glycosaminoglycan interactions to inhibit inflammation. Front Immunol. 2020;11:483. doi: 10.3389/fimmu.2020.00483

 

  1. Akiyama T, Yamamoto T. Regulation of early lymphocyte development via mRNA decay catalyzed by the CCR4-NOT Complex. Front Immunol. 2021;12:715675. doi: 10.3389/fimmu.2021.715675

 

  1. Su Z, Chen L, Niu Q, Yang B, Huang Z. Association of gene polymorphisms in CXC chemokine receptor 5 with rheumatoid arthritis susceptibility. Iran J Allergy Asthma Immunol. 2022;21(5):537-548. doi: 10.18502/ijaai.v21i5.11041

 

  1. Shi J, Hou S, Fang Q, et al. PD-1 controls follicular T helper cell positioning and function. Immunity. 2018;49(2):264- 274.e264. doi: 10.1016/j.immuni.2018.06.012

 

  1. Kazanietz MG, Durando M, Cooke M. CXCL13 and its receptor CXCR5 in cancer: Inflammation, immune response, and beyond. Front Endocrinol (Lausanne). 2019;10:471. doi: 10.3389/fendo.2019.00471

 

  1. Shiels MS, Pfeiffer RM, Hildesheim A, et al. Circulating inflammation markers and prospective risk for lung cancer. J Natl Cancer Inst. 2013;105(24):1871-1880. doi: 10.1093/jnci/djt309

 

  1. Eide HA, Halvorsen AR, Sandhu V, et al. Non-small cell lung cancer is characterised by a distinct inflammatory signature in serum compared with chronic obstructive pulmonary disease. Clin Transl Immunol. 2016;5(11):e109. doi: 10.1038/cti.2016.65

 

  1. Hussain M, Adah D, Tariq M, et al. CXCL13/CXCR5 signaling axis in cancer. Life Sci. 2019;227:175-186. doi: 10.1016/j.lfs.2019.04.053

 

  1. Panse J, Friedrichs K, Marx A, et al. Chemokine CXCL13 is overexpressed in the tumour tissue and in the peripheral blood of breast cancer patients. Br J Cancer. 2008;99(6):930-938. doi: 10.1038/sj.bjc.6604621

 

  1. Singh S, Singh R, Sharma PK, et al. Serum CXCL13 positively correlates with prostatic disease, prostate-specific antigen and mediates prostate cancer cell invasion, integrin clustering and cell adhesion. Cancer Lett. 2009;283(1):29-35. doi: 10.1016/j.canlet.2009.03.022

 

  1. Ouchida T, Isoda Y, Nakamura T, 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

 

  1. Asano T, Suzuki H, Tanaka T, et al. C(3)Mab-3: A monoclonal antibody for mouse CC chemokine receptor 3 for flow cytometry. Monoclon Antib Immunodiagn Immunother. 2022;41(2):74-79. doi: 10.1089/mab.2021.0062

 

  1. Li G, Tanaka T, Suzuki H, Kaneko MK, Kato Y. Cx1Mab-1: A novel anti-mouse CXCR1 monoclonal antibody for flow cytometry. Monoclon Antib Immunodiagn Immunother. 2024;43:59-66. doi: 10.1089/mab.2023.0031

 

  1. Tanaka T, Nanamiya R, Takei J, et al. Development of anti-mouse CC chemokine receptor 8 monoclonal antibodies for flow cytometry. Monoclon Antib Immunodiagn Immunother. 2021;40(2):65-70. doi: 10.1089/mab.2021.0005

 

  1. Gu X, Li D, Wu P, et al. Revisiting the CXCL13/CXCR5 axis in the tumor microenvironment in the era of single-cell omics: Implications for immunotherapy. Cancer Lett. 2024;605:217278. doi: 10.1016/j.canlet.2024.217278

 

  1. Tanaka T, Li G, Asano T, et al. Development of a novel anti-mouse CCR2 monoclonal antibody (C(2)Mab-6) by N-terminal peptide immunization. Monoclon Antib Immunodiagn Immunother. 2022;41(2):80-86. doi: 10.1089/mab.2021.0063

 

  1. Asano T, Suzuki H, Goto N, et al. Establishment of novel anti-mouse CCR3 monoclonal antibodies (C3 mab-6 and C3Mab-7) by N-terminal peptide immunization. Monoclon Antib Immunodiagn Immunother. 2022;41(2):94-100. doi: 10.1089/mab.2021.0065

 

  1. Takei J, Suzuki H, Asano T, et al. Development of a novel anti-mouse CCR4 monoclonal antibody (C4Mab-1) by N-terminal peptide immunization. Monoclon Antib Immunodiagn Immunother. 2022;41(2):87-93. doi: 10.1089/mab.2021.0064

 

  1. Asano T, Tanaka T, Suzuki H, et al. Development of a novel anti-mouse CCR6 monoclonal antibody (C6Mab-13) by N-terminal peptide immunization. Monoclon Antib Immunodiagn Immunother. 2022;41(6):343-349. doi: 10.1089/mab.2022.0021

 

  1. Kobayashi H, Asano T, Suzuki H, et al. Establishment of a sensitive monoclonal antibody against mouse CCR9 (C9Mab-24) for flow cytometry. Monoclon Antib Immunodiagn Immunother. 2023;42(1):15-21. doi: 10.1089/mab.2022.0032

 

  1. Kitamura K, Suzuki H, Kaneko MK, Kato Y. Cx6Mab-1: A novel anti-mouse CXCR6 monoclonal antibody established by N-terminal peptide immunization. Monoclon Antib Immunodiagn Immunother. 2022;41(3):133-141. doi: 10.1089/mab.2022.0010

 

  1. Satofuka H, Suzuki H, Tanaka T, et al. A Novel Anti-mouse CCR7 Monoclonal Antibody, C7Mab-7, Demonstrates High Sensitivity in Flow Cytometry, Western Blot, and Immunohistochemistry. [Preprints]; 2024.

 

  1. Available from: https://www.biolegend.com/ja-jp/products/ purified-anti-mouse-cd185-cxcr5-antibody-8454 [Last accessed on 2024 May 10].

 

  1. Hsieh CH, Jian CZ, Lin LI, et al. Potential role of CXCL13/ CXCR5 signaling in immune checkpoint inhibitor treatment in cancer. Cancers (Basel). 2022;14(2):294. doi: 10.3390/cancers14020294

 

  1. Crotty S. Follicular helper CD4 T cells (TFH). Annu Rev Immunol. 2011;29:621-663. doi: 10.1146/annurev-immunol-031210-101400

 

  1. Im SJ, Hashimoto M, Gerner MY, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature. 2016;537(7620):417-421. doi: 10.1038/nature19330

 

  1. Brummelman J, Mazza EMC, Alvisi G, et al. High-dimensional single cell analysis identifies stem-like cytotoxic CD8(+) T cells infiltrating human tumors. J Exp Med. 2018;215(10):2520-2535. doi: 10.1084/jem.20180684

 

  1. He R, Hou S, Liu C, et al. Follicular CXCR5- expressing CD8(+) T cells curtail chronic viral infection. Nature. 2016;537(7620):412-428. doi: 10.1038/nature19317

 

  1. Kallies A, Zehn D, Utzschneider DT. Precursor exhausted T cells: Key to successful immunotherapy? Nat Rev Immunol. 2020;20(2):128-136. doi: 10.1038/s41577-019-0223-7

 

  1. Thommen DS, Koelzer VH, Herzig P, et al. A transcriptionally and functionally distinct PD-1(+) CD8(+) T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade. Nat Med. 2018;24(7):994-1004. doi: 10.1038/s41591-018-0057-z

 

  1. De Chaisemartin L, Goc J, Damotte D, et al. Characterization of chemokines and adhesion molecules associated with T cell presence in tertiary lymphoid structures in human lung cancer. Cancer Res. 2011;71(20):6391-6399. doi: 10.1158/0008-5472.Can-11-0952

 

  1. Mitkin NA, Hook CD, Schwartz AM, et al. p53-dependent expression of CXCR5 chemokine receptor in MCF-7 breast cancer cells. Sci Rep. 2015;5:9330. doi: 10.1038/srep09330

 

  1. Mitkin NA, Muratova AM, Sharonov GV, et al. p63 and p73 repress CXCR5 chemokine receptor gene expression in p53-deficient MCF-7 breast cancer cells during genotoxic stress. Biochim Biophys Acta Gene Regul Mech. 2017;1860(12):1169-1178. doi: 10.1016/j.bbagrm.2017.10.003

 

  1. Takei J, Kaneko MK, Ohishi T, et al. A defucosylated antiCD44 monoclonal antibody 5mG2af exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma. Oncol Rep. 2020;44(5):1949-1960. doi: 10.3892/or.2020.7735

 

  1. Hosono H, Takei J, Ohishi T, et al. AntiEGFR monoclonal antibody 134mG2a exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma. Int J Mol Med. 2020;46(4):1443-1452. doi: 10.3892/ijmm.2020.4700

 

  1. Itai S, Ohishi T, Kaneko MK, et al. Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma. Oncotarget. 2018;9(32):22480-22497. doi: 10.18632/oncotarget.25132

 

  1. Liu T, Liu Y, Liu CX, Jiang YM. CXCL13 is elevated in inflammatory bowel disease in mice and humans and is implicated in disease pathogenesis. Front Immunol. 2022;13:997862. doi: 10.3389/fimmu.2022.997862
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