Abstract
Interleukin-5 (IL-5) binding to interleukin-5 receptor subunit alpha (IL-5Rα) increases the number of eosinophils and enhances eosinophil activity. This leads to eosinophil tissue infiltration and damage to the lungs, ultimately resulting in exacerbation of asthma. Antibodies that block IL-5 binding to IL-5Rα are thought to play an important role in advanced asthma. Currently, key methods used to screen for targeted drugs are Surface Plasmon Resonance which is costly and anti-proliferation assays which are tedious and have a low signal-to-noise ratio. Here we describe a Fluorescence Activated Cell Sorting (FACS) assay, based on human embryonic kidney (HEK)-293 cells with stable expression of IL-5Rα and the cytokine receptor common subunit beta (CSF2RB). Cells co-expressing IL-5Rα and CSF2RB had a 16% increase in the ability to bind IL-5 compared to cells expressing only IL-5Rα. The optimal concentration of IL-5 for the FACS assay was 0.1 µg/mL. The established FACS was used to screen anti IL-5 nanobodies and hybridoma supernatants for candidate antibodies that block the IL-5/IL-5α interaction. When compared to anti-proliferation assays, this method saved up to 90% of the assay time, offering the advantage of rapidity and accuracy in vitro. The assay described here provides a novel approach for rapid screening of IL-5/IL-5Rα blocking antibodies in vitro to accelerate the development of drugs for asthma.
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To, T., S. Stanojevic, G. Moores, A. S. Gershon, E. D. Bateman, A. A. Cruz, and L. P. Boulet (2012) Global asthma prevalence in adults: findings from the cross-sectional world health survey. BMC Public Health 12: 104. (Erratum published 2021, BMC Public Health 21: 1809)
Kolbeck, R., A. Kozhich, M. Koike, L. Peng, C. K. Andersson, M. M. Damschroder, J. L. Reed, R. Woods, W. W. Dall’acqua, G. L. Stephens, J. S. Erjefalt, L. Bjermer, A. A. Humbles, D. Gossage, H. Wu, P. A. Kiener, G. L. Spitalny, C. R. Mackay, N. A. Molfino, and A. J. Coyle (2010) MEDI-563, a humanized anti-IL-5 receptor alpha mAb with enhanced antibody-dependent cell-mediated cytotoxicity function. J. Allergy Clin. Immunol. 125: 1344–1353.e2.
Price, D. B., A. Rigazio, J. D. Campbell, E. R. Bleecker, C. J. Corrigan, M. Thomas, S. E. Wenzel, A. M. Wilson, M. B. Small, G. Gopalan, V. L. Ashton, A. Burden, E. V. Hillyer, M. Kerkhof, and I. D. Pavord (2015) Blood eosinophil count and prospective annual asthma disease burden: a UK cohort study. Lancet Respir. Med. 3: 849–858.
Hearn, A. P., B. D. Kent, and D. J. Jackson (2020) Biologic treatment options for severe asthma. Curr. Opin. Immunol. 66: 151–160.
Molfino, N. A., D. Gossage, R. Kolbeck, J. M. Parker, and G. P. Geba (2012) Molecular and clinical rationale for therapeutic targeting of interleukin-5 and its receptor. Clin. Exp. Allergy 42: 712–737.
Dougan, M., G. Dranoff, and S. K. Dougan (2019) GM-CSF, IL-3, and IL-5 family of cytokines: regulators of inflammation. Immunity 50: 796–811.
Nagase, H., S. Ueki, and S. Fujieda (2020) The roles of IL-5 and anti-IL-5 treatment in eosinophilic diseases: asthma, eosinophilic granulomatosis with polyangiitis, and eosinophilic chronic rhinosinusitis. Allergol. Int. 69: 178–186.
Yanagibashi, T., M. Satoh, Y. Nagai, M. Koike, and K. Takatsu (2017) Allergic diseases: from bench to clinic - contribution of the discovery of interleukin-5. Cytokine 98: 59–70.
Mita, S., S. Takaki, Y. Hitoshi, A. G. Rolink, A. Tominaga, N. Yamaguchi, and K. Takatsu (1991) Molecular characterization of the beta chain of the murine interleukin 5 receptor. Int. Immunol. 3: 665–672.
Moon, B. G., T. Yoshida, M. Shiiba, K. Nakao, M. Katsuki, S. Takaki, and K. Takatsu (2001) Functional dissection of the cytoplasmic subregions of the interleukin-5 receptor alpha chain in growth and immunoglobulin G1 switch recombination of B cells. Immunology 102: 289–300.
Patino, E., A. Kotzsch, S. Saremba, J. Nickel, W. Schmitz, W. Sebald, and T. D. Mueller (2011) Structure analysis of the IL-5 ligand-receptor complex reveals a wrench-like architecture for IL-5Ra. Structure 19: 1864–1875.
Wen, T., B. J. Aronow, Y. Rochman, M. Rochman, K. Kc, P. J. Dexheimer, P. Putnam, V. Mukkada, H. Foote, K. Rehn, S. Darko, D. Douek, and M. E. Rothenberg (2019) Single-cell RNA sequencing identifies inflammatory tissue T cells in eosinophilic esophagitis. J. Clin. Invest. 129: 2014–2028.
Tamayev, R., S. Gupta, and A. Rubinstein (2017) Anti-IL5 monoclonal antibodies may provide benefit in asthmatics on chronic oral steroid therapy masking eosinophilia. Ann. Allergy Asthma Immunol. 119(5 Suppl): S62.
Laviolette, M., D. L. Gossage, G. Gauvreau, R. Leigh, R. Olivenstein, R. Katial, W. W. Busse, S. Wenzel, Y. Wu, V. Datta, R. Kolbeck, and N. A. Molfino (2013) Effects of benralizumab on airway eosinophils in asthmatic patients with sputum eosinophilia. J. Allergy Clin. Immunol. 132: 1086–1096.e5. (Erratum published 2014, J. Allergy Clin. Immunol. 133: 1232)
Van Heeke, G., K. Allosery, V. De Brabandere, T. De Smedt, L. Detalle, and A. de Fougerolles (2017) Nanobodies® as inhaled biotherapeutics for lung diseases. Pharmacol. Ther. 169: 47–56.
Maillet, A., L. Guilleminault, E. Lemarié, S. Lerondel, N. Azzopardi, J. Montharu, N. Congy-Jolivet, P. Reverdiau, B. Legrain, C. Parent, D. H. Douvin, J. Hureaux, Y. Courty, M. De Monte, P. Diot, G. Paintaud, A. Le Pape, H. Watier, and N. Heuzé-Vourc’h (2011) The airways, a novel route for delivering monoclonal antibodies to treat lung tumors. Pharm. Res. 28: 2147–2156.
Haworth, R., M. Boyle, P. Edwards, R. Gupta, R. Fagg, G. Karantabias, and M. Price (2021) Preclinical safety assessment of 2 inhaled single-domain antibodies in the cynomolgus monkey. Toxicol. Pathol. 49: 296–314.
Tavernier, J., R. Devos, J. Van der Heyden, G. Hauquier, R. Bauden, I. Fache, E. Kawashima, J. Vandekerckhove, R. Contreras, and W. Fiers (1989) Expression of human and murine interleukin-5 in eukaryotic systems. DNA 8: 491–501.
Brown, P. M., M. P. Scheid, G. P. Oneil, P. C. Tagari, and D. W. Nicholson (1995) A single-step purification of biologically active recombinant human interleukin-5 from a baculovirus expression system. Protein Expr. Purif. 6: 63–71.
Dickason, R. R., M. M. Huston, and D. P. Huston (1994) Enhanced detection of human IL-5 in biological fluids utilizing murine monoclonal antibodies which delineate distinct neutralizing epitopes. Cytokine 6: 647–656.
Fu, Z., C. Yu, L. Wang, K. Gao, G. Xu, W. Wang, J. Cao, and J. Wang (2018) Development of a robust reporter gene based assay for the bioactivity determination of IL-5-targeted therapeutic antibodies. J. Pharm. Biomed. Anal. 148: 280–287.
Yen, J. J., Y. C. Hsieh, C. L. Yen, C. C. Chang, S. Lin, and H. F. Yang-Yen (1995) Restoring the apoptosis suppression response to IL-5 confers on erythroleukemic cells a phenotype of IL-5-dependent growth. J. Immunol. 154: 2144–2152.
Mita, S., Y. Hosoya, I. Kubota, T. Nishihara, T. Honjo, T. Takahashi, and K. Takatsu (1989) Rapid methods for purification of human recombinant interleukin-5 (IL-5) using the anti-murine IL-5 antibody-coupled immunoaffinity column. J. Immunol. Methods 125: 233–241.
Azuma, C., T. Tanabe, M. Konishi, T. Kinashi, T. Noma, F. Matsuda, Y. Yaoita, K. Takatsu, L. Hammarström, C. I. Smith, E. Severinson, and T. Honjo (1986) Cloning of cDNA for human T-cell replacing factor (interleukin-5) and comparison with the murine homologue. Nucleic Acids Res. 14: 9149–9158.
Jain, N. K., S. Barkowski-Clark, R. Altman, K. Johnson, F. Sun, J. Zmuda, C. Y. Liu, A. Kita, R. Schulz, A. Neill, R. Ballinger, R. Patel, J. Liu, A. Mpanda, B. Huta, H. Chiou, W. Voegtli, and T. Panavas (2017) A high density CHO-S transient transfection system: comparison of ExpiCHO and Expi293. Protein Expr. Purif. 134: 38–46.
Kupczyk, M. and P. Kuna (2018) Benralizumab: an anti-IL-5 receptor a monoclonal antibody in the treatment of asthma. Immunotherapy 10: 349–359.
Ryu, J., E. J. Kim, J. K. Kim, T. H. Park, B. G. Kim, and H. J. Jeong (2022) Development of a CHO cell line for stable production of recombinant antibodies against human MMP9. BMC Biotechnol. 22: 8.
Liang, Z., W. Jiao, L. Wang, Y. Chen, D. Li, Z. Zhang, Z. Zhang, Y. Liang, and H. Niu (2022) CYP27A1 inhibits proliferation and migration of clear cell renal cell carcinoma via activation of LXRs/ABCA1. Exp. Cell Res. 419: 113279.
Pelaia, C., G. Paoletti, F. Puggioni, F. Racca, G. Pelaia, G. W. Canonica, and E. Heffler (2019) Interleukin-5 in the pathophysiology of severe asthma. Front. Physiol. 10: 1514.
Schleich, F., G. Brusselle, R. Louis, O. Vandenplas, A. Michils, C. Pilette, R. Peche, M. Manise, and G. Joos (2014) Heterogeneity of phenotypes in severe asthmatics. The Belgian Severe Asthma Registry (BSAR). Respir. Med. 108: 1723–1732.
Murata, Y., S. Takaki, M. Migita, Y. Kikuchi, A. Tominaga, and K. Takatsu (1992) Molecular cloning and expression of the human interleukin 5 receptor. J. Exp. Med. 175: 341–351.
Hercus, T. R., U. Dhagat, W. L. T. Kan, S. E. Broughton, T. L. Nero, M. Perugini, J. J. Sandow, R. J. D’Andrea, P. G. Ekert, T. Hughes, M. W. Parker, and A. F. Lopez (2013) Signalling by the βc family of cytokines. Cytokine Growth Factor Rev. 24: 189–201.
Scheide-Noeth, J. P., M. Rosen, D. Baumstark, H. Dietz, and T. D. Mueller (2019) Structural basis of interleukin-5 inhibition by the small cyclic peptide AF17121. J. Mol. Biol. 431: 714–731.
Acknowledgements
The authors would like to express their gratitude to Sanyou Bio Inc., China, for the protein provided. This work was carried out with the support of “National first-class discipline program of Light Industry Technology and Engineering (LITE2018-24) and Zhuhai Science and Technology Innovation Project (ZH2201700220000ZPWC)”.
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Designed and carried out the experiments to construct stably cell lines: Shijie Li and Fei Han; Designed and carried out the FACS assay: Shijie Li, Chang Liu, and Weiyan Dai; Protein expression and purification: Wenfeng Ke; Drafted the work and revised: Shijie Li and Eric Fordjour; Final approval of the version to be published: Shijie Li, Yongqi Chen, Yankun Yang, and Zhonghu Bai.
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Li, S., Han, F., Liu, C. et al. Rapid Screen of IL-5/IL-5Rα Blocking Antibodies in the HEK293-IL-5Rα-CSF2RB Transfected Cell Line. Biotechnol Bioproc E 28, 612–622 (2023). https://doi.org/10.1007/s12257-022-0315-2
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DOI: https://doi.org/10.1007/s12257-022-0315-2