Insect venoms can cause severe allergic reactions, including anaphylaxis, in sensitized individuals. In this study, we aim at preventing anaphylaxis mediated by the most abundant and dominant honeybee venom allergen phospholipase A2 (Api m 1) by blocking its interaction with allergic patient IgE. Therefore, we characterize selected Api m 1-specific nanobodies and identify two high-affinity binders with non-overlapping epitopes. Crystal structures of Api m 1/nanobody complexes reveal diametrically opposed epitopes, one of which involves the active site of Api m 1. Based on this background, we develop mono- and bispecific nanobody-human IgG1 Fc, which exhibits pronounced blocking of IgE binding and effector cell activation in blood samples from honeybee venom allergic patients and reduces systemic reactions in a mouse model of allergen-induced anaphylaxis. This work provides a rationale for using nanobody-based inhibitors to prevent Api m 1-mediated anaphylaxis in honeybee venom allergy.

Background: Molecular technologies have paved the way to improved understanding of allergic diseases in many ways, ranging from molecular allergens to tailor-made tools for analytic, diagnostic, and therapeutic purposes. In particular, the progress in molecular targeting opens a variety of opportunities for improving protection and treatment of allergies and anaphylaxis. Methods: This review summarizes the state-of-the-art and the feasibility of applying molecular tools for exemplary applications within insect venom allergy and anaphylaxis. Results: In recent years, novel technologies have been established and applied in the development of antibodies offering advantages compared to current approaches. These antibodies might overcome current limitations and provide novel opportunities for treatment of Hymenoptera venom allergy. Hence, novel targets, molecular architectures, and forms of application of antibodies may provide a benefit for the allergic patient. Conclusion: Recent approaches give a first glimpse of the future possibilities of targeting approaches in a complex system such as allergic diseases. It has become clear that the simplicity of state-of-the-art antibody technologies will both broaden and deepen the scope of applications in allergology. Cite this as Ballegaard A-SR, Jensen BK, Aagaard JB, Miehe M, Spillner E. Molecular targeting in the context of insect venom allergy: targets and perspectives. Allergo J Int. 2026;35:8-14 https://doi.org/10.1007/s40629-025-00355-7

ABSTRACT Background IgE is the central driver of allergic responses. Prior studies have defined the conformation of the IgE Fc fragment bound to the FcεRIα ectodomain and the dynamic properties of the IgE Fc. It remains unknown, how these prior studies translate to the complex of a full antibody including the Fab arms with the receptor. Methods For structural analysis, crystallography, cryo‐EM and negative stain EM (ns‐EM) were combined. IgE variants were analyzed by mediator release and CD23 binding assays. Results An ensemble of 10 cryo‐EM structures of the full‐size IgE FcεRIα complex was obtained revealing that the receptor bound IgE adopts a pronounced T‐like conformation. Either Fab arm may rotate up to 40°. Two additional conformations with different arrangements of the Fab arms were captured in ns‐EM. The introduction of additional flexibility into the Fab‐Fc hinge does not compromise the biological activity of IgE, suggesting that the observed conformations of the IgE Fab‐Fc hinge exhibit equivalent biological function. Comparison of the full IgE receptor complex with recent cryo‐EM structures of the intact receptor reveals that FcεRI conformations differ markedly by the orientation of the ectodomain. Hence, our ensemble of IgE FcεRIα structures including the Fab arms enabled critical evaluation of FcεRI conformations. Conclusion Our data reveal the architecture of a full‐size IgE antibody bound to its receptor and a new layer of dynamics in FcεRIα bound IgE on top of the well‐established IgE Fc conformations. Development of novel anti‐IgE therapeutics may take into account these properties of FcεRIα bound IgE.

Molecular technologies have paved the way to improved understanding of allergic diseases in many ways, ranging from molecular allergens to tailor-made tools for analytical, diagnostic, and therapeutic purposes. Engineering of such molecules has become a mainstay in most biotechnical and biomedical areas. A not so new kid on the block is the nanobody, a single-domain antibody obtained from primarily camelid species. Despite their large promise and potential, it took nanobodies a long time to also enter the stage in allergology. This review summarizes the state of the art and the feasibility of engineering nanobody-based tools for applications in allergology. In recent years, nanobodies with specificity for allergens have been increasingly generated. In parallel, their molecular engineering has enabled the development of derivatives that offer many advantages compared to standard antibody approaches. Hence, different application forms of nanobody-based molecules have been developed and reported in proof-of-concept studies. Recent studies give a first glimpse of the future possibilities of nanobody technologies in a complex system such as allergic diseases. It has become clear that the simplicity of the approaches as compared to regular antibody technologies will both broaden and deepen the scope of applications in allergology.

PDF - 94K, Supplementary Methods.

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.

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.

Immunoglobulin E is a mammal specific antibody isotype supporting the immune response against parasites and venoms, but also a driver of allergic responses. Prior studies have defined the conformation of the IgE Fc fragment bound to the cell surface receptor FcεRIα and the dynamic properties of the IgE Fc. It remains unknown, how these prior studies translate to the complex of a full antibody including the Fab arms with the receptor. Here we show that in a cryo-EM structure of the IgE FcεRIα complex, IgE adopts a T-like conformation where the antigen binding Fab arms may be parallel to the cell membrane. Two additional conformations are captured in negative stain EM (ns-EM) where the arrangements of the Fab arms differ from the cryo-EM conformation. Small angle scattering data favors the FcεRIα bound IgE conformation observed by cryo-EM, but the major IgE conformation observed by ns-EM possibly may also occur. In all observed conformations of FcεRIα bound IgE, one Fab arm is fixed relative to the IgE Fc moiety whereas the second Fab may alternate its position. Introduction of flexibility in the Fab-Fc hinge diminishes the biological activity of IgE demonstrating a functional role for the observed defined Fab-Fc hinge conformations. Our data show the organization of a full size antibody on its receptor and reveal a new layer of dynamics in FcεRIα bound IgE on top of the well established spectrum of IgE Fc conformations. Development of novel anti-IgE therapeutics may take into account these distinct FcεRIα bound IgE conformations. Significance statement IgE represents a canonical antibody isotype and is a key molecule for the allergic immune response to environmental triggers driven by mast cells and basophils. The requirements for efficient mediation of IgE’s effects are not fully understood. Here we elucidate the structure of the entire IgE in complex with its high affinity receptor and identify two clearly distinct and dominant conformations, in which one of the Fab arms is fixed relative to the Fc domains. Enforcing IgE flexibility impacts the biological function with potential consequences for the allergic response. This unique behavior makes IgE different from all other isotypes and its understanding sheds light on the allergenic activation of the immune response.

Breast cancer (BC) treatment has traditionally been challenging due to tumor heterogeneity. Bispecific antibodies (bsAbs) offer a promising approach for overcoming these challenges by targeting multiple specific epitopes. In the current study, we designed a new bsAb against the most common BC cell surface proteins (SPs). To achieve this, we analyzed RNA-sequencing data to identify differentially expressed genes, which were further evaluated using Gene Ontology enrichment, Hidden Markov Models, clinical trial data, and survival analysis to identify druggable gene-encoding cell SPs. Based on these analyses, we constructed and expressed a bsAb targeting the mucin 1 (MUC1) and epidermal growth factor receptor (EGFR) proteins, which are the dominant druggable gene-encoding cell SPs in BC. The recombinant anti-MUC1×EGFR bsAb demonstrated efficient production and high specificity for MUC1 and EGFR + cell lines and BC tissue. Furthermore, the bsAb significantly reduced the proliferation and migration of BC cells. Our results suggested that simultaneous targeting with bsAbs could be a promising targeted therapy for improving the overall efficacy of BC treatment.

PDF - 210K, Can225IgG heavy and light chain amino acid sequences.

Hintergrund: Molekulare Technologien haben in vielerlei Hinsicht den Weg zu einem besseren Verständnis allergischer Erkrankungen geebnet, angefangen bei rekombinanten Allergenen bis hin zu maßgeschneiderten Werkzeugen für analytische, diagnostische und therapeutische Zwecke. Die Entwicklung solcher Moleküle ist inzwischen ein fester Bestandteil vieler biotechnologischer und biomedizinischer Bereiche. Ein nicht mehr ganz neues Konzept ist das der sogenannten Nanobodies, Einzeldomänen-Antikörper, die ihren primären Ursprung in Kamelen haben. Trotz ihres Potenzials hat der Einzug der Nanobody-Technologien in die Allergologie vergleichsweise lange gedauert. Methoden: In dieser Übersicht werden der aktuelle Stand und die Machbarkeit der Entwicklung von Nanobody-basierten Werkzeugen für Anwendungen in der Allergologie zusammengefasst. Ergebnisse: In den letzten Jahren wurden zunehmend Nanobodies mit Spezifität für Allergene entwickelt. Parallel dazu hat ihre molekulare Evolution die Erzeugung von unterschiedlichen Nanobody-Formaten ermöglicht, die gegenüber herkömmlichen Antikörpern deutliche Vorteile bieten. Auf dieser Grundlage wurden unterschiedliche Anwendungsformen von Nanobody-basierten Molekülen etabliert und in Machbarkeitsstudien vorgestellt. Diskussion: Aktuelle Studien geben einen ersten Einblick in die zukünftigen Einsatzmöglichkeiten von Nanobody-Technologien in einem komplexen System wie dem der allergischen Erkrankungen. Dabei wird deutlich, dass die Einfachheit dieser Ansätze im Vergleich zu herkömmlichen Antikörper-Technologien den Anwendungsbereich in der Allergologie sowohl erweitern als auch vertiefen wird. Zitierweise: Aagaard JB, Ballegaard A-SR, Andersen PO, Spillner E. Molecular engineering of nanobodies as tools in allergology: diagnostics and beyond. Allergo J Int 2023;32:240-50 https://doi.org/10.1007/s40629-023-00261-w

PDF - 446K, Figure S1. Purification of can225IgG from cell culture supernatant is efficient with Protein G, but not with Protein A. Figure S2. Elution of can225IgG from Protein G columns at a low pH does not affect integrity of the antibody. Figure S3. Binding of can225IgG to the cell lines CF33, CF41, TLM-1 and BT474 in FACS. Figure S4. Can225IgG shows a slightly lower binding affinity towards canine EGFR, compared with human EGFR. Figure S5. Staining of EGFR+ cell line and canine mammary carcinoma section by can225IgG.

Allergies have become a major challenge for our health systems, affecting a constantly increasing population, which suffer either from life threatening anaphylaxis or chronic inflammatory diseases. Thus, a better understanding of the pathophysiological principles underlying the different allergic disorders is of paramount importance for an optimized disease management, which includes the development of both novel therapies targeting key factors of allergic inflammation, and efficient strategies of prevention. To foster collaborative research efforts in the field of allergy and immunology, the German Societies of Immunology (DGfI) and of Allergy and Clinical Immunology (DGAKI) have established the task force ,Allergy & Immunology’ to join forces exploring the immune mechanisms of allergic endotypes and to gain new insights as to how these translate into clinical phenotypes and could be addressed by therapeutic measures. Here, we point out recent, seminal findings in important fields of allergy research comprising innate and humoral immunity, type-2 inflammation, environmental impacts, and the most current achievements in our understanding of inducing tolerance. In addition, we outline urgent questions and notable challenges ahead, which need to be tackled to shed more light on how immune signals evolve into allergic diseases and chronic inflammation, and to identify new solutions of either reversing their course or prevent their development.

The first publications on cross-reactive carbohydrate epitopes as targets of IgE antibodies to pollen protein appeared over 30 years ago, but in general they were not relevant to pollen symptoms. By contrast, the recently described IgE specific for alpha-gal which is cross reactive over many mammalian molecules, are causally related to two forms of anaphylaxis. Identification of protein allergens is based on the species of origin, assuming that each amino acid chain reflects a specific gene (www.allergen.org). By contrast, the blood group oligosaccharides including alpha-gal are produced by a sequence of enzymes in the endoplasmic reticulum and the golgi. The absence of alpha-gal in the primates reflects a de-functioning mutation in the gene for alpha 1, 3 galactosyl transferase, that occurred ∼20 million years ago. The incorporation of glycan nomenclature into the IUIS allergen nomenclature database raised many problems, because of the contrast with protein epitopes. Oligosaccharide epitopes can be on lipids with an O-linkage, and this linkage is relevant to lipid particles entering the circulation as chylomicrons or LDL. The terminology used for describing oligosaccharides is complex, but most epitopes can be illustrated as stick diagram and are relatively small. The new addition to the IUIS/WHO database is jointly funded by AAAAI and EAACI, and includes both structures and evidence about the clinical relevance of the IgE antibodies.

Immune responses to N-glycan structures from allergens and parasites are often associated with pronounced, high affinity IgE reactivities. Cross-reactive carbohydrate determinants (CCDs) are constituted by modified N-glycan core structures and represent the most frequently recognized epitopes in allergic immune responses. Although recently accepted as potentially allergenic epitopes, the biological and clinical relevance as well as structural and functional characteristics of CCD-specific antibodies remain elusive. In order to gain structural insights into the recognition of CCDs, two specific antibody fragments were isolated from a leporid immune repertoire library and converted into human/leporid IgE and IgG formats. The antibody formats were assessed by ELISA and surface plasmon resonance, structural and functional analyses were performed by X-ray crystallography, mediator release, and ELIFAB assays. The recombinant IgE exhibited highly specific interactions with different types of CCDs on numerous CCD-carrying glycoproteins. Crystal structures of two CCD-specific antibodies, one of which in complex with a CCD-derived disaccharide emphasize that mechanisms of core glycan epitope recognition are as specific as those governing protein epitope recognition. The rIgE triggered immediate cellular responses via FcεRI cross-linking and mediated facilitated antigen presentation by binding of IgE/antigen complexes to CD23, a process that also could be blocked by IgG of allergic patients. Our study provides evidence for the relevance of N-glycan recognition in T H 2 responses and corroborates that IgE and IgG antibodies to ubiquitous carbohydrate epitopes can be equivalent to those directed against proteinaceous epitopes with implications for diagnostic and immunotherapeutic concepts.

For the canonical antibody isotypes, antigen recognition is driven by the variable regions of both heavy and light chains. In contrast, single-domain antibodies or nanobodies (nb) are the antigen-binding moiety of heavy chain only antibodies occurring in camelid species and cartilaginous fish.1, 2 Their small-size, high-yield production, and stability render nanobodies versatile building blocks for the development of binders and unorthodox activatory or inhibitory multi-domain derivatives.3 In the allergological context, single nanobodies were used as anti-IgE molecules and as binding moiety for allergens.4, 5 Our aim was to generate allergen-specific nanobodies and establish a nanobody-based IgE format in the context of hymenoptera venom allergy (Figure 1A,B). In venom-allergic patients, the IgE response is typically directed to a set of major and minor allergens.6 The best-characterized and most abundant honeybee venom (HBV) allergens are phospholipase A2 (Api m 1) and hyaluronidase (Api m 2) (Figure S1A). Therefore, native Api m 1 and recombinant Api m 2 were used for llama immunization (Figure S1B,C). Immune libraries were generated from peripheral blood mononuclear cells. Enrichment by phage display and immunoreactivity of individual phage clones was shown by ELISA (Figure S2). After expression and purification from bacterial supernatant, the nanobodies showed the expected molecular masses in SDS-PAGE and immunoreactivity to their target in ELISA (Figure S3A–C). Exemplary nanobodies against Api m 1 (AM1-1 and AM1-3) and against Api m 2 (AM2-A1 and AM2-C2) were then converted into homodimeric IgE formats by fusion to IgE CH2-4 domains. After purification from supernatant of stably transfected HEK293 cells, SDS-PAGE and immunoblotting analyses corroborated proper dimerization of the nb-IgE (Figure 1C, Figure S4). Expression yields of 10–20 mg/L pointed at a favorable performance of the nb-IgE in mammalian hosts as compared to the limited yields often observed for entire IgE antibodies.7 The nb-IgEs remained reactive to both their particular target allergen and the FcεRIα in ELISA (Figure 1D). The antibodies exhibited sIgE reactivity in Euroline assays to HBV and individual allergens without cross-reactivity to other HBV or yellow jacket venom allergens (Figure 2A,B). Furthermore, individual nb-IgE clones were applied on the ImmunoCAP test system. Concentration-dependent sIgE reactivity was detected for Api m 1, Api m 2, and HBV (Figure 2C) corresponding well to the total IgE levels. The sIgE reactivity against Api m 1 was found highly comparable to that of HBV. In contrast, a clearly reduced IgE reactivity to HBV compared with the component Api m 2 pointed toward a lower sensitivity of HBV for patients with sensitization to Api m 2 possibly due to a limited accessibility of immunoreactive Api m 2 in the test. Furthermore, an oligoclonal artificial human serum was established by combining the 4 nb-IgE against Api m 1 and Api m 2 in equimolar concentration. Assessment of sIgE reactivity verified the added sIgE levels for HBV, Api m 1, and Api m 2 (Figure 2D). Notably, the reduced sIgE level for HBV reflected the reduced reactivity of the individual Api m 2-specific nb-IgE to HBV. Hence, artificial sera comprising molecularly defined IgE surrogates of adjustable concentration might replace human sera in applications such as assessment of diagnostic test systems, round-robin tests, and diagnostic and therapeutic extracts, but also mechanistical analyses in basic research. In summary, we have established nanobodies as a toolbox for the generation of important downstream formats like the novel nb-IgE. This platform also paves the way for the generation of other formats including blocking IgG formats, and hence, has the potential to provide advanced tools for diagnostics, functional analyses, and interventional studies. Gratefully acknowledged is the excellent technical assistance by Nanna Breum Nielsen, Britta Dorn, and Manuel Schulze-Dasbeck. Figure 1 A and B was created with BioRender.com. This study was supported by the Novo Nordisc Foundation, grant NNF19OC0058484, and the Independent Research Foundation Denmark, grant 9041-00291A. A patent application covering part of the manuscript has been filed by JBA, CS, MM, and ES. The other authors have no financial conflict of interest and nothing to disclose. Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.