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A robust and stable reporter gene bioassay for anti-IgE antibodies

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Abstract

Immunoglobin E (IgE)-related allergy constitutes a high proportion in allergic diseases. The production of specific IgE is key to evoking serial cascades and pathological reactions. Thus, targeting IgE is a different therapeutic approach from symptomatic treatments. Monoclonal antibodies (mAbs) against IgE were developed and a humanized antibody, omalizumab, was approved by five countries. It could inhibit the binding of IgE with epsilon receptor I of crystallizable fragment (FcεRI), thus preventing anaphylactic reactions. However, no bioactivity assay, which is the critical quality attribute and should thoroughly reflect the clinical mechanism, has been established to date. In commercial lot release, only the enzyme-linked immunosorbent assay (ELISA) method was applied, which only reflects the binding of omalizumab to IgE but not the subsequent reaction. In scientific research works, human FcεRI-transfected RBL-2H3 cells were used to indicate degranulation based on the detection of β-hexosaminidase. Nevertheless, this method needs much work to stabilize the response and, hence, is not suitable for routine usage in commercial production and control of antibodies. To evaluate the bioactivity of anti-IgE antibodies including omalizumab using a simple assay that reflects the following mechanism of actions (MOA) after binding, we established an RBL-2H3 cell line transfected with both the α subunit of human FcεRI and nuclear factor-activated T cell (NFAT) response elements, the latter is conjugated with a luciferase gene, which could shed luminescence when substrates exist. The method was proven to possess good specificity, accuracy, linearity, and precision and may be utilized as a supplement to anti-IgE antibody bioactivity assays in terms of development, lot release, stability, and comparability studies.

The mechanism sketch of reporter gene assay for bioactivity determination of anti-IgE antibodies by RBL-2H3/FcεRIα/NFAT-Luc cells (left) and representative curves generated by the reporter gene assay (right).

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References

  1. Kalesnikoff J, Huber M, Lam V, Damen JE, Zhang J, Siraganian RP, et al. Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity. 2001;14:801–11.

    PubMed  CAS  Google Scholar 

  2. Walker SMM, Phelan K, Lasserson TJ, Walters EH. Anti-IgE for chronic asthma in adults and children. Cochrane Database Syst Rev. 2006;19(2):CD003559. https://doi.org/10.1002/14651858.CD003559.pub3.

  3. Douglass JA, O’Hehir RE. Diagnosis, treatment and prevention of allergic disease: the basics. Med J Aust. 2006;185(4):228–33.

    PubMed  Google Scholar 

  4. Kawakami T, Blank U. From IgE to omalizumab. J Immunol. 2016;197(11):4187–92.

    PubMed  PubMed Central  CAS  Google Scholar 

  5. Oettgen HC. Fifty years later: emerging functions of IgE antibodies in host defense, immune regulation, and allergic diseases. J Allergy Clin Immunol. 2016;137(6):1631–45.

    PubMed  PubMed Central  CAS  Google Scholar 

  6. Baena-Cagnani CE. The global burden of asthma and allergic diseases: the challenge for the new century. Curr Allergy Asthma Rep. 2001;1(4):297–8.

    PubMed  CAS  Google Scholar 

  7. Braman SS. The global burden of asthma. Chest. 2006;130(1 Suppl):4S–12S.

    PubMed  Google Scholar 

  8. Ehteshami-Afshar S, Fitzgerald JM, Doyle-Waters MM, Sadatsafavi M. The global economic burden of asthma and chronic obstructive pulmonary disease. Int J Tuberc Lung Dis. 2016;20(1):11–23.

    PubMed  CAS  Google Scholar 

  9. Cruz AA, Stelmach R, Ponte EV. Asthma prevalence and severity in low-resource communities. Curr Opin Allergy Clin Immunol. 2017;17(3):188–93.

    PubMed  Google Scholar 

  10. Anto JM, Pinart M, Akdis M, Auffray C, Bachert C, Basagana X, et al. Understanding the complexity of IgE-related phenotypes from childhood to young adulthood: a Mechanisms of the Development of Allergy (MeDALL) seminar. J Allergy Clin Immunol. 2012;129(4):943–54 e4.

    PubMed  Google Scholar 

  11. Hoffmann A, Vieths S, Haustein D. Biologic allergen assay for in vivo test allergens with an in vitro model of the murine type I reaction. J Allergy Clin Immunol. 1997;99(2):227–32.

    PubMed  CAS  Google Scholar 

  12. Vogel L, Luttkopf D, Hatahet L, Haustein D, Vieths S. Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system. Allergy. 2005;60(8):1021–8.

    PubMed  CAS  Google Scholar 

  13. Navines-Ferrer A, Serrano-Candelas E, Molina-Molina GJ, Martin M. IgE-related chronic diseases and anti-IgE-based treatments. J Immunol Res. 2016;2016:8163803.

    PubMed  PubMed Central  Google Scholar 

  14. Platts-Mills TAE, Schuyler AJ, Erwin EA, Commins SP, Woodfolk JA. IgE in the diagnosis and treatment of allergic disease. J Allergy Clin Immunol. 2016;137(6):1662–70.

    PubMed  PubMed Central  CAS  Google Scholar 

  15. Khodoun MV, Kucuk ZY, Strait RT, Krishnamurthy D, Janek K, Lewkowich I, et al. Rapid polyclonal desensitization with antibodies to IgE and FcepsilonRIalpha. J Allergy Clin Immunol. 2013;131(6):1555–64.

    PubMed  PubMed Central  CAS  Google Scholar 

  16. MacGinnitie AJ, Rachid R, Gragg H, Little SV, Lakin P, Cianferoni A, et al. Omalizumab facilitates rapid oral desensitization for peanut allergy. J Allergy Clin Immunol. 2017;139(3):873–81 e8.

    PubMed  CAS  Google Scholar 

  17. Holgate S, Buhl R, Bousquet J, Smith N, Panahloo Z, Jimenez P. The use of omalizumab in the treatment of severe allergic asthma: a clinical experience update. Respir Med. 2009;103(8):1098–113.

    PubMed  Google Scholar 

  18. D’Amato G, Stanziola A, Sanduzzi A, Liccardi G, Salzillo A, Vitale C, et al. Treating severe allergic asthma with anti-IgE monoclonal antibody (omalizumab): a review. Multidiscip Respir Med. 2014;9(1):23. https://doi.org/10.1186/2049-6958-9-23.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Palmer GW. RBL cells expressing human FcεRI are a sensitive tool for exploring functional IgE-allergen interactions: studies with sera from peanut-sensitive patients. J Immunol Methods. 2003;274(1):37–45.

    PubMed  Google Scholar 

  20. Hoffmann HJ, Santos AF, Mayorga C, Nopp A, Eberlein B, Ferrer M, et al. The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease. Allergy. 2015;70(11):1393–405.

    PubMed  CAS  Google Scholar 

  21. Takagi K, Nakamura R, Teshima R, Sawada J. Application of human FcεRI a-chain-transfected RBL-2H3 cells for estimation of active serum IgE. Biol Pharm Bull. 2003;26(2):252–5.

    PubMed  CAS  Google Scholar 

  22. Rice LV, Bax HJ, Russell LJ, Barrett VJ, Walton SE, Deakin AM, et al. Characterization of selective calcium-release activated calcium channel blockers in mast cells and T-cells from human, rat, mouse and guinea-pig preparations. Eur J Pharmacol. 2013;704(1–3):49–57.

    PubMed  CAS  Google Scholar 

  23. Sun R, Yang Y, Ran X, Yang T. Calcium influx of mast cells is inhibited by aptamers targeting the first extracellular domain of Orai1. PLoS One. 2016;11(7):e0158223.

    PubMed  PubMed Central  Google Scholar 

  24. Tomita S, Matsuda A, Nishinami S, Kurita R, Shiraki K. One-step identification of antibody degradation pathways using fluorescence signatures generated by cross-reactive DNA-based arrays. Anal Chem. 2017;89(15):7818–22.

    PubMed  CAS  Google Scholar 

  25. Nakamura R, Uchida Y, Higuchi M, Nakamura R, Tsuge I, Urisu A, et al. A convenient and sensitive allergy test: IgE crosslinking-induced luciferase expression in cultured mast cells. Allergy. 2010;65(10):1266–73.

    PubMed  PubMed Central  CAS  Google Scholar 

  26. Gauvreau GM, Arm JP, Boulet LP, Leigh R, Cockcroft DW, Davis BE, et al. Efficacy and safety of multiple doses of QGE031 (ligelizumab) versus omalizumab and placebo in inhibiting allergen-induced early asthmatic responses. J Allergy Clin Immunol. 2016;138(4):1051–9.

    PubMed  CAS  Google Scholar 

  27. Casale TB, Bernstein IL, Busse WW, CF LF, Tinkelman DG, Stoltz RR, et al. Use of an anti-IgE humanized monoclonal antibody in ragweed-induced allergic rhinitis. J Allergy Clin Immunol. 1997;100(1):110–21.

    PubMed  CAS  Google Scholar 

  28. Adelroth E, Rak S, Haahtela T, Aasand G, Rosenhall L, Zetterstrom O, et al. Recombinant humanized mAb-E25, an anti-IgE mAb, in birch pollen-induced seasonal allergic rhinitis. J Allergy Clin Immunol. 2000;106(2):253–9.

    PubMed  CAS  Google Scholar 

  29. Presta L, Shields R, O’Connell L, Lahr S, Porteri J, Goman C, et al. The binding site on human immunoglobulin E for its high affinity receptor. J Biol Chem. 1994;269(42):26368–73.

    PubMed  CAS  Google Scholar 

  30. Larson D, Mitre E. Histamine release and surface CD200R1 staining as sensitive methods for assessing murine mast cell activation. J Immunol Methods. 2012;379(1–2):15–22.

    PubMed  PubMed Central  CAS  Google Scholar 

  31. Huang L, Pi J, Wu J, Zhou H, Cai J, Li T, et al. A rapid and sensitive assay based on particle analysis for cell degranulation detection in basophils and mast cells. Pharmacol Res. 2016;111:374–83.

    PubMed  CAS  Google Scholar 

  32. Nyborg AC, Zacco A, Ettinger R, Jack Borrok M, Zhu J, Martin T, et al. Development of an antibody that neutralizes soluble IgE and eliminates IgE expressing B cells. Cell Mol Immunol. 2016;13(3):391–400.

    PubMed  CAS  Google Scholar 

  33. Ho HP, Lau PM, Kwok HC, Wu SY, Gao M, Cheung AK, et al. Allergen screening bioassays: recent developments in lab-on-a-chip and lab-on-a-disc systems. Bioanalysis. 2014;6(14):2005–18.

    PubMed  CAS  Google Scholar 

  34. Knipping K, van Roest M, Kruijssen L, Smits M, Teunis M, Cox L, et al. Intra- and inter-laboratory validation of an innovative huFcepsilonRIalpha-RBL-2H3 degranulation assay for in vitro allergenicity assessment of whey hydrolysates. Toxicol in Vitro. 2016;33:29–34.

    PubMed  CAS  Google Scholar 

  35. Blott EJ, Griffiths GM. Secretory lysosomes. Nat Rev Mol Cell Biol. 2002;3(2):122–31.

    PubMed  CAS  Google Scholar 

  36. Kleine-Tebbe J, Erdmann S, Knol EF, MacGlashan DW Jr, Poulsen LK, Gibbs BF. Diagnostic tests based on human basophils: potentials, pitfalls and perspectives. Int Arch Allergy Immunol. 2006;141(1):79–90.

    PubMed  Google Scholar 

  37. Chirumbolo S. Basophil activation test in allergy: time for an update? Int Arch Allergy Immunol. 2012;158(2):99–114.

    PubMed  Google Scholar 

  38. Anna N, Johansson SGO, Adédoyin J, Ankerst J, Palmqvist M, Oman H. After 6 years with Xolair; a 3-year withdrawal follow-up. Allergy. 2010;65(1):56–60.

    Google Scholar 

  39. Holgate ST. New strategies with anti-IgE in allergic diseases. World Allergy Organ J. 2014;7(1):17.

    PubMed  PubMed Central  Google Scholar 

  40. Incorvaia C, Mauro M, Russello M, Formigoni C, Riario-Sforza GG, Ridolo E. Omalizumab, an anti-immunoglobulin E antibody: state of the art. Drug Des Devel Ther. 2014;8:197–207.

    PubMed  PubMed Central  CAS  Google Scholar 

  41. Wang L, Yu C, Wang J. Development of reporter gene assays to determine the bioactivity of biopharmaceuticals. Biotechnol Adv. 2019:107466.

  42. Wang L, Xu GL, Gao K, Wilkinson J, Zhang F, Yu L, et al. Development of a robust reporter-based assay for the bioactivity determination of anti-VEGF therapeutic antibodies. J Pharm Biomed Anal. 2016;125:212–8.

    PubMed  CAS  Google Scholar 

  43. Yang Y, Zhou Y, Yu L, Li X, Shi X, Qin X, et al. A novel reporter gene assay for recombinant human erythropoietin (rHuEPO) pharmaceutical products. J Pharm Biomed Anal. 2014;100:316–21.

    PubMed  CAS  Google Scholar 

  44. Yu L, Shi X, Han C, Rao C, Wang J. A rapid reporter assay for recombinant human brain natriuretic peptide (rhBNP) by GloSensor technology. J Pharm Anal. 2018;8(5):297–301.

    PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by the National Science and Technology Major Projects for Major New Drugs Innovation and Development (Grant number: 2018ZX09736-008).

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Author notes

  1. Sha Guo and Chuanfei Yu contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, No. 31, Huatuo Road, Daxing District, Beijing, 102629, China

    Sha Guo, Chuanfei Yu, Feng Zhang, Junxia Cao & Lan Wang

  2. Shanghai Taiyin Biotechnology Co., Ltd, No. 781 Cailun Road, Zhangjiang Hi-tech Park, Shanghai, 201203, China

    Yanchao Wang & Chen Zheng

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  1. Sha Guo
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Correspondence to Lan Wang.

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Guo, S., Yu, C., Wang, Y. et al. A robust and stable reporter gene bioassay for anti-IgE antibodies. Anal Bioanal Chem 412, 1901–1914 (2020). https://doi.org/10.1007/s00216-020-02442-w

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  • DOI: https://doi.org/10.1007/s00216-020-02442-w

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