Skip to main content
SpringerLink
Log in

Extract-Shaped Immune Repertoires as Source for Nanobody-Based Human IgE in Grass Pollen Allergy

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The presence of allergen-specific IgE in serum is a biomarker for allergic disease. Specific IgE antibodies for research and diagnostics, however, remain scarce. In contrast to prototypic antibodies, camelid species have evolved single domains as moiety for antigen recognition. These so-called nanobodies represent a versatile platform for the development of diagnostic and therapeutic approaches. In this study, we aimed for generating nanobodies and derived IgE formats from an extract-shaped immune repertoire. Timothy grass pollen represents a complex, but well-defined mixture of individual allergens. Therefore, a repertoire library from a timothy grass pollen extract immunised llama was established. The selection by phage display yielded 3 nanobodies with immunoreactivity to the extract. IgE-like nanobody-based human IgE (nb-hIgE) antibodies were produced in mammalian cells and assessed in different immunoassays and commercial platforms. Immunoblotting and diagnostic ImmunoCap analysis of single timothy grass pollen allergens identified the major allergens Phl p 6 and Phl p 4 as targets. Assessment of immunoreactivity further documented significant molecular cross-reactivity with pollen extract of different grass species and variant presence of allergens within extracts of Pooideae grasses. In summary, our study shows that extract-based immunisation enables the generation of allergen-specific nanobodies and derived nb-hIgE formats linking nanobody technologies with allergological applications.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

Data used and analysed in this study are available from the authors on reasonable request.

References

  1. Finkelman, F. D., Boyce, J. A., Vercelli, D., & Rothenberg, M. E. (2010). Key advances in mechanisms of asthma, allergy, and immunology in 2009. The Journal of Allergy and Clinical Immunology, 125, 312–318.

    Article  CAS  PubMed  Google Scholar 

  2. Gould, H. J., & Sutton, B. J. (2008). IgE in allergy and asthma today. Nature reviews. Immunology, 8, 205–217.

    Article  CAS  PubMed  Google Scholar 

  3. Chang, T. W. (2000). The pharmacological basis of anti-IgE therapy. Nature Biotechnology, 18, 157–162.

    Article  CAS  PubMed  Google Scholar 

  4. Clement, M. J., Fortune, A., Phalipon, A., Marcel-Peyre, V., Simenel, C., Imberty, A., Delepierre, M., & Mulard, L. A. (2006). Toward a better understanding of the basis of the molecular mimicry of polysaccharide antigens by peptides: The example of Shigella flexneri 5a. Journal of Biological Chemistry, 281, 2317–2332.

    Article  CAS  PubMed  Google Scholar 

  5. Andersson, K., & Lidholm, J. (2003). Characteristics and immunobiology of grass pollen allergens. International Archives of Allergy and Immunology, 130, 87–107.

    Article  CAS  PubMed  Google Scholar 

  6. Dewitt, A. M., Andersson, K., Peltre, G., & Lidholm, J. (2006). Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5. Clinical and Experimental Allergy, 36, 77–86.

    Article  CAS  PubMed  Google Scholar 

  7. Platts-Mills, T. A., Hilger, C., Jappe, U., Hage, M., Gadermaier, G., Spillner, E., Jonas, L., Keshavarz, B., Aalberse, R. C., Van Ree, R., Goodman, R. E., & Pomes, A. (2021). Carbohydrate epitopes currently recognized as targets for IgE antibodies. Allergy, 76, 2383.

    Article  CAS  PubMed  Google Scholar 

  8. Plum, M., Tjerrild, L., Raiber, T., Bantleon, F., Bantleon, S., Miehe, M., Jabs, F., Seismann, H., Mobs, C., Pfutzner, W., Jakob, T., Andersen, G. R., & Spillner, E. (2022). Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in TH 2 responses. Allergy, 78, 121–130.

    Article  PubMed  Google Scholar 

  9. Aalberse, R. C., Koshte, V., & Clemens, J. G. (1981). Immunoglobulin E antibodies that crossreact with vegetable foods, pollen, and Hymenoptera venom. The Journal of Allergy and Clinical Immunology, 68, 356–364.

    Article  CAS  PubMed  Google Scholar 

  10. Kohler, J., Blank, S., Muller, S., Bantleon, F., Frick, M., Huss-Marp, J., Lidholm, J., Spillner, E., & Jakob, T. (2014). Component resolution reveals additional major allergens in patients with honeybee venom allergy. Journal of Allergy and Clinical Immunology, 133, 1383–1389.

    Article  CAS  PubMed  Google Scholar 

  11. Akdis, C. A., & Akdis, M. (2014). Mechanisms of immune tolerance to allergens: Role of IL-10 and Tregs. The Journal of Clinical Investigation, 124, 4678–4680.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Boonpiyathad, T., Meyer, N., Moniuszko, M., Sokolowska, M., Eljaszewicz, A., Wirz, O. F., Tomasiak-Lozowska, M. M., Bodzenta-Lukaszyk, A., Ruxrungtham, K., & van de Veen, W. (2017). High-dose bee venom exposure induces similar tolerogenic B-cell responses in allergic patients and healthy beekeepers. Allergy, 72, 407–415.

    Article  CAS  PubMed  Google Scholar 

  13. Meiler, F., Zumkehr, J., Klunker, S., Ruckert, B., Akdis, C. A., & Akdis, M. (2008). In vivo switch to IL-10-secreting T regulatory cells in high dose allergen exposure. Journal of Experimental Medicine, 205, 2887–2898.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Shamji, M., & Durham, S. (2017). Mechanisms of allergen immunotherapy for inhaled allergens and predictive biomarkers. The Journal of Allergy and Clinical Immunology, 140, 1485–1498.

    Article  CAS  PubMed  Google Scholar 

  15. Shamji, M. H., Ljorring, C., Francis, J. N., Calderon, M. A., Larche, M., Kimber, I., Frew, A. J., Ipsen, H., Lund, K., Wurtzen, P. A., & Durham, S. R. (2012). Functional rather than immunoreactive levels of IgG4 correlate closely with clinical response to grass pollen immunotherapy. Allergy, 67, 217–226.

    Article  CAS  PubMed  Google Scholar 

  16. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Songa, E. B., Bendahman, N., & Hamers, R. (1993). Naturally occurring antibodies devoid of light chains. Nature, 363, 446–448.

    Article  CAS  PubMed  Google Scholar 

  17. Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T., & Winter, G. (1989). Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature, 341, 544–546.

    Article  CAS  PubMed  Google Scholar 

  18. Konning, D., Zielonka, S., Grzeschik, J., Empting, M., Valldorf, B., Krah, S., Schroter, C., Sellmann, C., Hock, B., & Kolmar, H. (2016). Camelid and shark single domain antibodies: Structural features and therapeutic potential. Current Opinion in Structural Biology, 45, 10–16.

    Article  PubMed  Google Scholar 

  19. Zavrtanik, U., Lukan, J., Loris, R., Lah, J., & Hadzi, S. (2018). Structural basis of epitope recognition by heavy-chain camelid antibodies. Journal of Molecular Biology, 430, 4369–4386.

    Article  CAS  PubMed  Google Scholar 

  20. Detalle, L., Stohr, T., Palomo, C., Piedra, P. A., Gilbert, B. E., Mas, V., Millar, A., Power, U. F., Stortelers, C., Allosery, K., Melero, J. A., & Depla, E. (2015). Generation and characterization of ALX-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection. Antimicrobial agents and chemotherapy, 60, 6–13.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Fahy, J. V., Cockcroft, D. W., Boulet, L. P., Wong, H. H., Deschesnes, F., Davis, E. E., Ruppel, J., Su, J. Q., & Adelman, D. C. (1999). Effect of aerosolized anti-IgE (E25) on airway responses to inhaled allergen in asthmatic subjects. American Journal of Respiratory and Critical Care Medicine, 160, 1023–1027.

    Article  CAS  PubMed  Google Scholar 

  22. Braren, I., Blank, S., Seismann, H., Deckers, S., Ollert, M., Grunwald, T., & Spillner, E. (2007). Generation of human monoclonal allergen-specific IgE and IgG antibodies from synthetic antibody libraries. Clinical Chemistry, 53, 837–844.

    Article  CAS  PubMed  Google Scholar 

  23. Hecker, J., Diethers, A., Etzold, S., Seismann, H., Michel, Y., Plum, M., Bredehorst, R., Blank, S., Braren, I., & Spillner, E. (2011). Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the timothy grass major allergen Phl p 5a. Molecular Immunology, 48, 1236–1244.

    Article  CAS  PubMed  Google Scholar 

  24. Hecker, J., Diethers, A., Schulz, D., Sabri, A., Plum, M., Michel, Y., Mempel, M., Ollert, M., Jakob, T., Blank, S., Braren, I., & Spillner, E. (2012). An IgE epitope of Bet v 1 and fagales PR10 proteins as defined by a human monoclonal IgE. Allergy, 67, 1530–1537.

    CAS  PubMed  Google Scholar 

  25. Plum, M., Michel, Y., Wallach, K., Raiber, T., Blank, S., Bantleon, F. I., Diethers, A., Greunke, K., Braren, I., Hackl, T., Meyer, B., & Spillner, E. (2011). Close-up of the immunogenic alpha1,3-galactose epitope as defined by a monoclonal chimeric immunoglobulin E and human serum using saturation transfer difference (STD) NMR. Journal of Biological Chemistry, 286, 43103–43111.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Aagaard, J. B., Sivelle, C., Fischer, M., Byskov, K., Laursen, N. S., Pfutzner, W., Jakob, T., Mobs, C., Miehe, M., & Spillner, E. (2022). Nanobody-based human antibody formats act as IgE surrogate in hymenoptera venom allergy. Allergy, 77, 2859–2862.

    Article  CAS  PubMed  Google Scholar 

  27. Jabs, F., Plum, M., Laursen, N. S., Jensen, R. K., Molgaard, B., Miehe, M., Mandolesi, M., Rauber, M. M., Pfutzner, W., Jakob, T., Mobs, C., Andersen, G. R., & Spillner, E. (2018). Trapping IgE in a closed conformation by mimicking CD23 binding prevents and disrupts FcepsilonRI interaction. Nature Communications, 9, 7.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Zettl, I., Ivanova, T., Zghaebi, M., Rutovskaya, M. V., Ellinger, I., Goryainova, O., Kollarova, J., Villazala-Merino, S., Lupinek, C., Weichwald, C., Drescher, A., Eckl-Dorna, J., Tillib, S. V., & Flicker, S. (2022). Generation of high affinity ICAM-1-specific nanobodies and evaluation of their suitability for allergy treatment. Frontiers in Immunology, 13, 1022418.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Akiba, H., Tamura, H., Kiyoshi, M., Yanaka, S., Sugase, K., Caaveiro, J. M. M., & Tsumoto, K. (2019). Structural and thermodynamic basis for the recognition of the substrate-binding cleft on hen egg lysozyme by a single-domain antibody. Scientific Reports, 9, 15481.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Chen, F., Ma, H., Li, Y., Wang, H., Samad, A., Zhou, J., Zhu, L., Zhang, Y., He, J., Fan, X., & Jin, T. (2019). Screening of nanobody specific for peanut major allergen Ara h 3 by phage display. Journal of Agricultural and Food Chemistry, 67, 11219–11229.

    Article  CAS  PubMed  Google Scholar 

  31. Hu, Y., Wu, S., Wang, Y., Lin, J., Sun, Y., Zhang, C., Gu, J., Yang, F., Lv, H., Ji, X., Zhang, Y., Muyldermans, S., & Wang, S. (2021). Unbiased immunization strategy yielding specific nanobodies against macadamia allergen of vicilin-like protein for immunoassay development. Journal of Agricultural and Food Chemistry, 69, 5178–5188.

    Article  CAS  PubMed  Google Scholar 

  32. Zettl, I., Ivanova, T., Strobl, M. R., Weichwald, C., Goryainova, O., Khan, E., Rutovskaya, M. V., Focke-Tejkl, M., Drescher, A., Bohle, B., Flicker, S., & Tillib, S. V. (2021). Isolation of nanobodies with potential to reduce patients IgE binding to Bet v 1 (68/100 characters). Allergy, 77, 1751–1760.

    Article  PubMed  Google Scholar 

  33. Matricardi, P. M., Kleine-Tebbe, J., Hoffmann, H. J., Valenta, R., Hilger, C., Hofmaier, S., Aalberse, R. C., Agache, I., Asero, R., Ballmer-Weber, B., Barber, D., Beyer, K., Biedermann, T., Bilo, M. B., Blank, S., Bohle, B., Bosshard, P. P., Breiteneder, H., Brough, H. A., et al. (2016). EAACI molecular allergology user’s guide. Pediatric Allergy and Immunology, 27(Suppl 23), 1–250.

    Article  PubMed  Google Scholar 

  34. Offermann, N., Plum, M., Hubner, U., Rathloff, K., Braren, I., Fooke, M., & Spillner, E. (2016). Human serum substitution by artificial sera of scalable allergen reactivity based on polyclonal antibodies and chimeras of human FcgammaRI and IgE domains. Allergy, 71, 1794–1799.

    Article  CAS  PubMed  Google Scholar 

  35. Wood, R. A., Segall, N., Ahlstedt, S., & Williams, P. B. (2007). Accuracy of IgE antibody laboratory results. Annals of Allergy, Asthma & Immunology, 99, 34–41.

    Article  Google Scholar 

  36. Popescu, F. D. (2014). Molecular biomarkers for grass pollen immunotherapy. World Journal of Methodology, 4, 26–45.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Vrtala, S., Fischer, S., Grote, M., Vangelista, L., Pastore, A., Sperr, W. R., Valent, P., Reichelt, R., Kraft, D., & Valenta, R. (1999). Molecular, immunological, and structural characterization of Phl p 6, a major allergen and P-particle-associated protein from Timothy grass (Phleum pratense) pollen. The Journal of Immunology, 163, 5489–5496.

    Article  CAS  PubMed  Google Scholar 

  38. Zafred, D., Nandy, A., Pump, L., Kahlert, H., & Keller, W. (2013). Crystal structure and immunologic characterization of the major grass pollen allergen Phl p 4. The Journal of Allergy and Clinical Immunology, 132, 696–703.

    Article  CAS  PubMed  Google Scholar 

  39. Westman, M., Aberg, K., Apostolovic, D., Lupinek, C., Gattinger, P., Mittermann, I., Andersson, N., Melen, E., Bergstrom, A., Anto, J. M., Bousquet, J., Valenta, R., Wickman, M., van Hage, M., Mechanisms for the Development of Allergies, c. (2020). Sensitization to grass pollen allergen molecules in a birth cohort-natural Phl p 4 as an early indicator of grass pollen allergy. The Journal of Allergy and Clinical Immunology, 145, 1174–1181.

    Article  CAS  PubMed  Google Scholar 

  40. Madeira, F., Pearce, M., Tivey, A. R. N., Basutkar, P., Lee, J., Edbali, O., Madhusoodanan, N., Kolesnikov, A., & Lopez, R. (2022). Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Research, 50, W1.

    Article  Google Scholar 

  41. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M., & Barton, G. J. (2009). Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics, 25, 1189–1191.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Gratefully acknowledged is the excellent technical assistance by Nanna Breum Nielsen. Figure 2A and 3A were created with BioRender.com. This study was supported by the the Novo Nordisk Foundation, grant NNF19OC0058484, and the Independent Research Foundation Denmark, grant 9041-00291A.

Author information

Authors and Affiliations

  1. Immunological Biotechnology, Department of Biological and Chemical Engineering, Aarhus University, Gustav Wieds Vej 10, 8000, Aarhus C, Denmark

    Josephine Baunvig Aagaard, Michaela Fischer, Julia Lober, Frederikke Bolbro Neumann, Dena Allahverdi, Coline Sivelle, Michaela Miehe & Edzard Spillner

Authors
  1. Josephine Baunvig Aagaard
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michaela Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Julia Lober
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Frederikke Bolbro Neumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dena Allahverdi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Coline Sivelle
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michaela Miehe
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Edzard Spillner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Edzard Spillner.

Ethics declarations

Conflict of interest

The authors declare no commercial or financial conflicts of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 539 kb)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Aagaard, J.B., Fischer, M., Lober, J. et al. Extract-Shaped Immune Repertoires as Source for Nanobody-Based Human IgE in Grass Pollen Allergy. Mol Biotechnol 65, 1518–1527 (2023). https://doi.org/10.1007/s12033-023-00664-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-023-00664-8

Keywords

Search

Navigation