This short review provides an introduction to the rapidly developing field of generation and utilization of “camel nanoantibodies” (or “nanobodies”). The term “nanoantibody” or “nanobody” was given to single-domain variable fragments of special type of antibodies that naturally exist (in addition to classical types of antibodies) in blood of Camelidae family animals and in some chondrichthyan fishes. The existence of very efficient technology of nanobody generation and some very useful characteristic features promise a big potential for their use in immunobiotechnology and medicine.

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.

In this work, IgG content and structures of antigen-binding domains and hinge regions of different IgG subtypes of Camelus bactrianus were analyzed in detail for the first time. Our data demonstrate that C. bactrianus contains a very large amount of heavy chain-only antibodies that can be used as a source of VHH domain-containing molecules. Despite some minor sequence differences identified in this study, C. bactrianus VHH domains possess principally the same unique features as those of C. dromedarius and the llama. These features are important for developing an efficient phage display-based anti- body selection technology. We conclude that C. bactrianus is a very suitable animal to raise an immune response that serves as a source to identify antigen-specific VHHs selected after phage display.

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.