Single-Domain Antibodies-Novel Tools to Study and Treat Allergies.

IgE-mediated allergies represent a major health problem in the modern world. Apart from allergen-specific immunotherapy (AIT), the only disease-modifying treatment, researchers focus on biologics that target different key molecules such as allergens, IgE, or type 2 cytokines to ameliorate allergic symptoms. Single-domain antibodies, or nanobodies, are the newcomers in biotherapeutics, and their huge potential is being investigated in various research fields since their discovery 30 years ago. While they are dominantly applied for theranostics of cancer and treatment of infectious diseases, nanobodies have become increasingly substantial in allergology over the last decade. In this review, we discuss the prerequisites that we consider to be important for generating useful nanobody-based drug candidates for treating allergies. We further summarize the available research data on nanobodies used as allergen monitoring and detection probes and for therapeutic approaches. We reflect on the limitations that have to be addressed during the development process, such as in vivo half-life and immunogenicity. Finally, we speculate about novel application formats for allergy treatment that might be available in the future.

Trimeric Bet v 1-specific nanobodies cause strong suppression of IgE binding.

Background: Around 20% of the population in Northern and Central Europe is affected by birch pollen allergy, with the major birch pollen allergen Bet v 1 as the main elicitor of allergic reactions. Together with its cross-reactive allergens from related trees and foods, Bet v 1 causes an impaired quality of life. Hence, new treatment strategies were elaborated, demonstrating the effectiveness of blocking IgG antibodies on Bet v 1-induced IgE-mediated reactions. A recent study provided evidence for the first time that Bet v 1-specific nanobodies reduce patients´ IgE binding to Bet v 1. In order to increase the potential to outcompete IgE recognition of Bet v 1 and to foster cross-reactivity and cross-protection, we developed Bet v 1-specific nanobody trimers and evaluated their capacity to suppress polyclonal IgE binding to corresponding allergens and allergen-induced basophil degranulation. Methods: Nanobody trimers were engineered by adding isoleucine zippers, thus enabling trimeric formation. Trimers were analyzed for their cross-reactivity, binding kinetics to Bet v 1, and related allergens, and patients' IgE inhibition potential. Finally, their efficacy to prevent basophil degranulation was investigated. Results: Trimers showed enhanced recognition of cross-reactive allergens and increased efficiency to reduce IgE-allergen binding compared to nanobody monomers. Furthermore, trimers displayed slow dissociation rates from allergens and suppressed allergen-induced mediator release. Conclusion: We generated high-affine nanobody trimers that target Bet v 1 and related allergens. Trimers blocked IgE-allergen interaction by competing with IgE for allergen binding. They inhibited IgE-mediated release of biological mediators, demonstrating a promising potential to prevent allergic reactions caused by Bet v 1 and relatives.

Antibody Conjugates Bispecific for Pollen Allergens and ICAM-1 with Potential to Prevent Epithelial Allergen Transmigration and Rhinovirus Infection.

Allergy and rhinovirus (RV) infections are major triggers for rhinitis and asthma, causing a socioeconomic burden. As RVs and allergens may act synergistically to promote airway inflammation, simultaneous treatment strategies for both causative agents would be innovative. We have previously identified the transmembrane glycoprotein intercellular adhesion molecule 1 (ICAM-1) as an anchor for antibody conjugates bispecific for ICAM-1 and Phleum pratense (Phl p) 2, a major grass pollen allergen, to block allergen transmigration through the epithelial barrier. Since ICAM-1 is a receptor for the major group RVs, we speculated that our bispecific antibody conjugates may protect against RV infection. Therefore, we created antibody conjugates bispecific for ICAM-1 and the major grass pollen allergen Phl p 5 and analyzed their capacity to affect allergen penetration and RV infection. Bispecific antibody conjugates significantly reduced the trans-epithelial migration of Phl p 5 and thus the basolateral Phl p 5 concentration and allergenic activity as determined by humanized rat basophilic leukemia cells and inhibited RV infection of cultured epithelial cells. A reduction in allergenic activity was obtained only through the prevention of allergen transmigration because the Phl p 5-specific IgG antibody did not block the allergen-IgE interaction. Our results indicate the potential of allergen/ICAM-1-specific antibody conjugates as a topical treatment strategy for allergy and RV infections.

Generation of high affinity ICAM-1-specific nanobodies and evaluation of their suitability for allergy treatment.

The nasal cavity is an important site of allergen entry. Hence, it represents an organ where trans-epithelial allergen penetration and subsequent IgE-mediated allergic inflammation can potentially be inhibited. Intercellular adhesion molecule 1 (ICAM-1) is highly expressed on the surface of respiratory epithelial cells in allergic patients. It was identified as a promising target to immobilize antibody conjugates bispecific for ICAM-1 and allergens and thereby block allergen entry. We have previously characterized a nanobody specific for the major birch pollen allergen Bet v 1 and here we report the generation and characterization of ICAM-1-specific nanobodies. Nanobodies were obtained from a camel immunized with ICAM-1 and a high affinity binder was selected after phage display (Nb44). Nb44 was expressed as recombinant protein containing HA- and His-tags in Escherichia coli (E.coli) and purified via affinity chromatography. SDS-PAGE and Western blot revealed a single band at approximately 20 kDa. Nb44 bound to recombinant ICAM-1 in ELISA, and to ICAM-1 expressed on the human bronchial epithelial cell line 16HBE14o- as determined by flow cytometry. Experiments conducted at 4°C and at 37°C, to mimic physiological conditions, yielded similar percentages (97.2 ± 1.2% and 96.7 ± 1.5% out of total live cells). To confirm and visualize binding, we performed immunofluorescence microscopy. While Texas Red Dextran was rapidly internalized Nb44 remained localized on the cell surface. Additionally, we determined the strength of Nb44 and ICAM-1 interaction using surface plasmon resonance (SPR). Nb44 bound ICAM-1 with high affinity (10-10 M) and had slow off-rates (10-4 s-1). In conclusion, our results showed that the selected ICAM-1-specific nanobody bound ICAM-1 with high affinity and was not internalized. Thus, it could be further used to engineer heterodimers with allergen-specific nanobodies in order to develop topical treatments of pollen allergy.

Isolation of nanobodies with potential to reduce patients' IgE binding to Bet v 1.

BACKGROUND: Recent studies showed that a single injection of human monoclonal allergen-specific IgG antibodies significantly reduced allergic symptoms in birch pollen-allergic patients. Since the production of full monoclonal antibodies in sufficient amounts is laborious and expensive, we sought to investigate if smaller recombinant allergen-specific antibody fragments, that is, nanobodies, have similar protective potential. For this purpose, nanobodies specific for Bet v 1, the major birch pollen allergen, were generated to evaluate their efficacy to inhibit IgE-mediated responses. METHODS: A cDNA-VHH library was constructed from a camel immunized with Bet v 1 and screened for Bet v 1 binders encoding sequences by phage display. Selected nanobodies were expressed, purified, and analyzed in regards of epitope-specificity and affinity to Bet v 1. Furthermore, cross-reactivity to Bet v 1-homologues from alder, hazel and apple, and their usefulness to inhibit IgE binding and allergen-induced basophil activation were investigated. RESULTS: We isolated three nanobodies that recognize Bet v 1 with high affinity and cross-react with Aln g 1 (alder) and Cor a 1 (hazel). Their epitopes were mapped to the alpha-helix at the C-terminus of Bet v 1. All nanobodies inhibited allergic patients' polyclonal IgE binding to Bet v 1, Aln g 1, and Cor a 1 and partially suppressed Bet v 1-induced basophil activation. CONCLUSION: We identified high-affinity Bet v 1-specific nanobodies that recognize an important IgE epitope and reduce allergen-induced basophil activation revealing the first proof that allergen-specific nanobodies are useful tools for future treatment of pollen allergy.

Nanobodies-Useful Tools for Allergy Treatment?

In the last decade single domain antibodies (nanobodies, VHH) qualified through their unique characteristics have emerged as accepted and even advantageous alternative to conventional antibodies and have shown great potential as diagnostic and therapeutic tools. Currently nanobodies find their main medical application area in the fields of oncology and neurodegenerative diseases. According to late-breaking information, nanobodies specific for coronavirus spikes have been generated these days to test their suitability as useful therapeutics for future outbreaks. Their superior properties such as chemical stability, high affinity to a broad spectrum of epitopes, low immunogenicity, ease of their generation, selection and production proved nanobodies also to be remarkable to investigate their efficacy for passive treatment of type I allergy, an exaggerated immune reaction to foreign antigens with increasing global prevalence.