Nanobody‐based human antibody formats act as <scp>IgE</scp> surrogate in hymenoptera venom allergy

Aagaard, Josephine Baunvig; Sivelle, Coline; Fischer, Michaela; Byskov, Kristina; Laursen, N.S.; Pfützner, Wolfgang; Jakob, Thilo; Möbs, Christian; Miehe, Michaela; Spillner, Edzard

doi:10.1111/all.15391

Cited by 7 publications

(4 citation statements)

References 7 publications

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“…Nasal and ocular epithelial cell surfaces are exposed to allergen quantities in the range of ng/day ( 55 ), thus stopping allergen penetration with rather low amounts of specific nanobodies topically applied to target organs of allergic rhinoconjunctivitis seems feasible. Increasing numbers of nanobodies specific for a variety of clinically relevant allergens are becoming available ( 27 – 30 , 56 , 57 ). It should therefore be possible to develop allergen/ICAM-1 nanobody constructs for diverse respiratory allergens that can be flexibly combined to protect patients against several different allergen sources.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, a nanobody capable of abrogating allergen-mediated basophil activation by mimicking CD23, the low affinity receptor for IgE, thus disrupting the bond between IgE and FcϵRI, was generated and suggested to act more efficiently than Omalizumab ( 58 ). Very recently, nanobody-IgE formats were described to act as surrogates in hymenoptera venom allergy and the authors speculated that formats consisting of blocking nanobodies and IgG antibodies could be used in interventional studies ( 56 ). These data are in line with our findings that allergen-specific or anti-IgE nanobodies could be effective for allergy treatment ( 38 , 57 , 58 ).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

et al. 2022

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“…In the context of allergic diseases, the use of blocking nanobodies for therapeutic purposes has been recently highlighted (101) and the generation of allergen-specific nanobodies has subsequently provided the initial corner stone for assessment of potential in allergy-related applications. Nanobodies have been generated against not only pollen allergens (97,102), and food allergens (103), but also venom allergens (104) and downstream formats like multimeric nanobodies and nanobody fusion proteins in the format of IgE have highlighted the ease of nanobody technologies.…”

Section: Molecular Targeting Of Epitopes For Therapymentioning

confidence: 99%

Potential and limitations of epitope mapping and molecular targeting in Hymenoptera venom allergy

Fernandes,

Spillner,

Jakob

2023

Front. Allergy

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Hymenoptera venom (HV) allergy can lead to life threatening conditions by specific IgE (sIgE)-mediated anaphylactic reactions. The knowledge about major allergens from venom of different clinically relevant species increased in the last decades, allowing the development of component-resolved diagnostics in which sIgE to single allergens is analysed. Despite these advances, the precise regions of the allergens that bind to IgE are only known for few HV allergens. The detailed characterization of IgE epitopes may provide valuable information to improve immunodiagnostic tests and to develop new therapeutic strategies using allergen-derived peptides or other targeted approaches. Epitope-resolved analysis is challenging, since the identification of conformational epitopes present in many allergens demands complex technologies for molecular analyses. Furthermore, functional analysis of the epitopeś interaction with their respective ligands is needed to distinguish epitopes that can activate the allergic immune response, from those that are recognized by irrelevant antibodies or T cell receptors from non-effector cells. In this review, we focus on the use of mapping and molecular targeting approaches for characterization of the epitopes of the major venom allergens of clinically relevant Hymenoptera species. The screening of the most relevant allergen peptides by epitope mapping could be helpful for the development of molecules that target major and immunodominant epitopes blocking the allergen induced cellular reactions as novel approach for the treatment of HV allergy.

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“…To overcome this challenge, gene-based approaches and various delivery modes of monoclonal antibodies such as inhalation or intranasal application have emerged as promising and advantageous options (27,29). In parallel, nanobodies have recently been discovered as potentially appropriate tools for allergy treatment (30)(31)(32)(33)(34)(35)(36). Their unique features such as their capacity to bind epitopes on their cognate antigens with a strong affinity that may not be accessible for monoclonal antibodies, their single domain organization, their easy and inexpensive generation in prokaryotic cells, render nanobodies valuable agents for food safety inspections but also for nanobody-mediated interventional studies (31,32,(36)(37)(38).…”

Section: Introductionmentioning

confidence: 99%

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

Bauernfeind,

Zettl,

Ivanova

et al. 2024

Front. Immunol.

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BackgroundAround 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.MethodsNanobody 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.ResultsTrimers 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.ConclusionWe 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.

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Nanobody‐based human antibody formats act as IgE surrogate in hymenoptera venom allergy

Cited by 7 publications

References 7 publications

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

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

Potential and limitations of epitope mapping and molecular targeting in Hymenoptera venom allergy

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

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