Publications - Immunological Biotechnology

Molekulares Design von Nanobodies als Werkzeuge in der Allergologie: Diagnostik und mehr

Aagaard, J. B., Ballegaard, A. R., Andersen, P. O., Spillner, E. — Sun, 01 Jan 2023 09:29:33 +0100

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

Romani Fernandez, L. G., Spillner, E., Jakob, T. — Sun, 01 Jan 2023 09:29:33 +0100

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.

Surfaceome Profiling Suggests Potential of Anti-MUC1×EGFR Bispecific Antibody for Breast Cancer Targeted Therapy

Pourjafar, M., Saidijam, M., Miehe, M., Najafi, R., Soleimani, M., Spillner, E. — Fri, 01 Sep 2023 09:29:33 +0200

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.

Molecular engineering of nanobodies as tools in allergology: diagnostics and beyond

Aagaard, J. B., Ballegaard, A. R., Andersen, P. O., Spillner, E. — Sun, 01 Oct 2023 09:29:33 +0200

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 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. Methods: This review summarizes the state of the art and the feasibility of engineering nanobody-based tools for applications in allergology. Results: 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. Discussion: 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.

A fully human monoclonal antibody isolated from beekeepers targets the immunodominant IgE epitope of Api m 10

Lund, A., Dorn, B., Jakob, T., Christensen, L. H., Jabs, F., Spillner, E. — Mon, 24 Jun 2024 09:29:33 +0200

Extract-shaped immune repertoires as source for nanobody-based human IgE in grass pollen allergy

Aagaard, J. B., Fischer, M. ., Lober, J., et al. — Wed, 25 Jan 2023 09:29:33 +0100

Construction, expression and functional characterization of an engineered antibody against tumor antigen MUC-1C

Pourjafar, M., Miehe, M., Najafi, R., Soleimani, M., Spillner, E. — Tue, 01 Nov 2022 09:29:33 +0100

Minibodies (single-chain Fv-CH3) are fusion proteins of a single-chain variable fragment (scFv) to the human IgG1 CH3 domain. They exhibit superior properties as compared to whole antibodies due to their smaller size and less complex composition, and also as compared to scFvs due to the two antigen-binding domains, for immunotherapy and imaging of various carcinomas including breast cancer. In the current study, efficient production of the recombinant anti-MUC-1 minibody for its dominant format (VH-VL) was obtained in the periplasmic space of the Escherichia coli BL21 (DE3) expression system. The active recombinant protein was successfully purified from soluble fraction. Functional assays presented the in vitro targeting properties and specificity of the expressed anti-MUC-1 HL minibody in the MUC-1 positive cell lines compared to normal cell.

Current research and unmet needs in allergy and immunology in Germany

Albrecht, M., Schaub, B., Gilles, S., et al. — Wed, 01 Jun 2022 09:29:33 +0200

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

Aagaard, J. B., Sivelle, C., Fischer, M. ., et al. — Thu, 01 Sep 2022 09:29:33 +0200

Carbohydrate Epitopes Currently Recognized as Targets for IgE Antibodies

Platts-Mills, T., Hilger, C., Jappe, U., et al. — Tue, 02 Mar 2021 09:29:33 +0100

The Honeybee Venom Major Allergen Api m 10 (Icarapin) and Its Role in Diagnostics and Treatment of Hymenoptera Venom Allergy

Jakob, T., Rauber, M. M., Perez-Riverol, A., Spillner, E., Blank, S. — Wed, 01 Jan 2020 09:29:33 +0100

Purpose of Review: In Hymenoptera venom allergy, the research focus has moved from whole venoms to individual allergenic molecules. Api m 10 (icarapin) has been described as a major allergen of honeybee venom (HBV) with potentially high relevance for diagnostics and therapy of venom allergy. Here, we review recent studies on Api m 10 characteristics as well as its role in component-resolved diagnostics and potential implications for venom-specific immunotherapy (VIT). Recent Findings: Api m 10 is a major allergen of low abundance in HBV. It is an obviously unstable protein of unknown function that exhibits homologs in other insect species. Despite its low abundance in HBV, 35 to 72% of HBV-allergic patients show relevant sensitization to this allergen. Api m 10 is a marker allergen for HBV sensitization, which in many cases can help to identify primary sensitization to HBV and, hence, to discriminate between genuine sensitization and cross-reactivity. Moreover, Api m 10 might support personalized risk stratification in VIT, as dominant sensitization to Api m 10 has been identified as risk factor for treatment failure. This might be of particular importance since Api m 10 is strongly underrepresented in some therapeutic preparations commonly used for VIT. Summary: Although the role of Api m 10 in HBV allergy and tolerance induction during VIT is not fully understood, it certainly is a useful tool to unravel primary sensitization and individual sensitization profiles in component-resolved diagnostics (CRD). Moreover, a potential of Api m 10 to contribute to personalized treatment strategies in HBV allergy is emerging.

Structure of intact IgE and the mechanism of ligelizumab revealed by electron microscopy

Jensen, R. K., Jabs, F., Miehe, M., et al. — Sat, 01 Aug 2020 09:29:33 +0200

Background: IgE is the central antibody isotype in TH2-biased immunity and allergic diseases. The structure of intact IgE and the impact of IgE-targeting molecules on IgE however remain elusive. In order to obtain insights into IgE biology and the clinical impact, we aimed for structure determination of IgE and the complex of IgE with the anti-IgE antibody ligelizumab. Methods: Structures of two distinct intact IgE with specificity for cross-reactive carbohydrate determinants and Der p 2 as well as complexes of ligelizumab-Fab with IgE and IgE Fc were assessed by negative stain electron microscopy and solution scattering. Inhibition of IgE binding and displacement of receptor-bound IgE were assessed using cellular assays, basophil activation testing and ELIFAB assays. Results: Our data reveal that the investigated IgE molecules share an overall rigid conformation. In contrast to the IgE Fc fragment, the IgE Fc in intact IgE is significantly less asymmetrically bent. The proximal and the distal Fabs are rigidly tethered to the Fc. Binding of ligelizumab to IgE in a 2:1 stoichiometry induces an extended and twofold symmetrical conformation of IgE, which retains a rigid Fab-Fc architecture. Analyses of effector cell activation revealed that ligelizumab inhibits IgE binding without displacing receptor-bound IgE. Together with an interference of CD23 binding, the data underline a functional activity similar to omalizumab. Conclusions: Our data reveal the first structures of intact IgE suggesting that the IgE Fab is fixed relative to the Fc. Furthermore, we provide a structural rationale for the inhibitory mechanism of ligelizumab.

The honey bee venom allergen Api m 10 displays one major IgE epitope, Api m 10160-174

Rauber, M., Roßbach, A., Jung, A., et al. — Wed, 01 Jul 2020 09:29:33 +0200

Work-related allergic asthma

Thu, 01 Aug 2019 09:29:33 +0200

In Silico Evaluation of the Binding Site of Fucosyltransferase 8 and First Attempts to Synthesize an Inhibitor with Drug-Like Properties

Strecker, C., Bärenfänger, M., Miehe, M., Spillner, E., Meyer, B. — Wed, 01 Jul 2020 09:29:33 +0200

Core fucosylation of N‐glycans is catalyzed by fucosyltransferase 8 and is associated with various types of cancer. Most reported fucosyltransferase inhibitors contain non‐drug‐like features, such as charged groups. New starting points for the development of inhibitors of fucosyltransferase 8 using a fragment‐based strategy are presented. Firstly, we discuss the potential of a new putative binding site of fucosyltransferase 8 that, according to a molecular dynamics (MD) simulation, is made accessible by a significant motion of the SH3 domain. This might enable the design of completely new inhibitor types for fucosyltransferase 8. Secondly, we have performed a docking study targeting the donor binding site of fucosyltransferase 8, and this yielded two fragments that were linked and trimmed in silico. The resulting ligand was synthesized. Saturation transfer difference (STD) NMR confirmed binding of the ligand featuring a pyrazole core that mimics the guanine moiety. This ligand represents the first low‐molecular‐weight compound for the development of inhibitors of fucosyltransferase 8 with drug‐like properties.

Inhibiting phosphatase SHIP-1 enhances suboptimal IgE-mediated activation of human blood basophils but inhibits IgE-mediated activation of cultured human mast cells

Rasmussen, P., Spillner, E., Hoffmann, H. J. — Sat, 01 Jun 2019 09:29:33 +0200

IgE-mediated activation of basophil granulocytes and mast cells follows a bell-shaped dose-response curve. The decreased activation at supraoptimal allergen stimulation is thought to be associated with SH2-containing inositol-5′-phosphatase 1 (SHIP-1). SHIP-1 phosphorylation is inversely related to IgE-mediated releasability of basophils. This study sought to clarify the regulatory role of SHIP-1 in degranulation of basophil granulocytes and mast cells by selective inhibition of the phosphatase function of SHIP-1with 3-α-aminocholestane (3-α-AC). Six grass pollen allergic patients, six non-responder patients and six cultured human primary mast cell lines were included. The effect of 3-α-AC (1–60 μM, 30 min, 37 °C) was analyzed at individual suboptimal, optimal and supra-optimal allergen concentrations. The activity, upregulation of CD63, measured at different conditions was compared to evaluate the maximal effect of selective SHIP-1 inhibition. Basophils of five non-responder patients were treated with 3-α-AC (10 μM, 30 min, 37 °C). At high concentrations (>60 μM) of 3-α-AC, cells appeared to enter apoptosis. The median reactivity increased from 27.1% to 44.9% CD63 ⁺ basophils at 10 μM of 3-α-AC and suboptimal allergen stimulation (p = 0.0153). There was no effect on blood basophils of 3-α-AC at optimal or supra-optimal allergen concentrations. In contrast, treatment with more than 6 μM 3-α-AC significantly inhibited mast cell reactivity. 10 μM 3-α-AC reduced median reactivity from 32.85% to 16.5% CD63+ mast cells (p = 0.0465). Treatment with 3-α-AC did not increase response of basophils of non-responder patients. Modulating blood basophils with 3-α-AC enhanced reactivity only at suboptimal allergen concentration, and basophils from non-responders did not regain responsiveness to IgE stimulation. 3-α-AC inhibited the IgE response of mast cells in a dose dependent manner.

Characterization of the honeybee venom proteins C1q-like protein and PVF1 and their allergenic potential

Russkamp, D., Van Vaerenbergh, M., Etzold, S., et al. — Wed, 01 Aug 2018 09:29:33 +0200

AllergoOncology: Generating a canine anti-cancer IgE against the epidermal growth factor receptor

Fazekas-Singer, J., Singer, J., Ilieva, K. M. ., et al. — Mon, 01 Jan 2018 09:29:33 +0100

N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Pedersen, C. T., Loke, I., Lorentzen, A., et al. — Tue, 01 Aug 2017 09:29:33 +0200

Venoms of Neotropical wasps lack cross-reactive carbohydrate determinants enabling reliable protein-based specific IgE determination

Riverol, A. P., Miehe, M., Jabs, F., et al. — Tue, 01 May 2018 09:29:33 +0200

Phospholipase A1-based cross-reactivity among venoms of clinically relevant Hymenoptera from Neotropical and temperate regions

Perez Riverol, A., Romani Fernandes, L. G. ., Musacchio Lasa, A. ., et al. — Mon, 01 Jan 2018 09:29:33 +0100

Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics.

Component resolved diagnostics for Hymenoptera venom allergy

Jakob, T., Müller, U., Helbling, A., Spillner, E. — Sun, 01 Jan 2017 09:29:33 +0100

Purpose of review Component-resolved diagnostics makes use of defined allergen molecules to analyse IgE-mediated sensitizations at a molecular level. Here, we review recent studies on the use of component-resolved diagnostics in the field of Hymenoptera venom allergy (HVA) and discuss its benefits and limitations. Recent findings Component resolution in HVA has moved from single molecules to panels of allergens. Detection of specific immunoglobulin E (sIgE) to marker and cross-reactive venom allergens has been reported to facilitate the discrimination between primary sensitization and cross-reactivity and thus, to provide a better rationale for prescribing venom immunotherapy (VIT), particularly in patients sensitized to both honeybee and vespid venom. Characterization of IgE reactivity to a broad panel of venom allergens has allowed the identification of different sensitization profiles that in honeybee venom allergy were associated with increased risks for side effects or treatment failure of VIT. In contrast, component resolution so far has failed to provide reliable markers for the discrimination of sensitizations to venoms of different members of Vespidae. Summary Component-resolved diagnostics allows a better understanding of the complexity of sensitization and cross-reactivities in HVA. In addition, the enhanced resolution and precision may allow identification of biomarkers, which can be used for risk stratification in VIT. Knowledge about the molecular composition of different therapeutic preparations may enable the selection of appropriate preparations for VIT according to individual sensitization profiles, an approach consistent with the goals of personalized medicine.

AllergoOncology - the Impact of Allergy in Oncology. EAACI Position Paper.

Jensen-Jarolim, E., Bax, H., Bianchini, R., et al. — Sun, 01 Jan 2017 09:29:33 +0100

Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both antitumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE-mediated immune responses against cancer, to derive novel insights into more effective treatments. Coincident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state-of-the-art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intratumoral innate allergy effector cells and mediators, IgE-mediated tumour antigen cross-presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the crosstalk between allergic response and cancer is paving the way for new avenues of treatment.

Trapping IgE in a closed conformation by mimicking CD23 binding prevents and disrupts FcεRI interaction

Jabs, F., Plum, M., Laursen, N. S., et al. — Tue, 02 Jan 2018 09:29:33 +0100

Anti-IgE therapeutics interfere with the ability of IgE to bind to its receptors on effector cells. Here we report the crystal structure of an anti-IgE single-domain antibody in complex with an IgE Fc fragment, revealing how the antibody inhibits interactions between IgE and the two receptors FcϵRI and CD23. The epitope overlaps only slightly with the FcϵRI-binding site but significantly with the CD23-binding site. Solution scattering studies of the IgE Fc reveal that antibody binding induces a half-bent conformation in between the well-known bent and extended IgE Fc conformations. The antibody acts as functional homolog of CD23 and induces a closed conformation of IgE Fc incompatible with FcϵRI binding. Notably the antibody displaces IgE from both CD23 and FcϵRI, and abrogates allergen-mediated basophil activation and facilitated allergen binding. The inhibitory mechanism might facilitate strategies for the future development of anti-IgE therapeutics for treatment of allergic diseases.

Recombinant allergens as a major step in molecular allergology:

Spillner, E. — Fri, 01 Jan 2016 09:29:33 +0100

Comparing sensitivity of Hymenoptera allergen components on different diagnostic assay systems

Jakob, T., Spillner, E. — Wed, 01 Mar 2017 09:29:33 +0100

Diagnostics in hymenoptera venom allergy: Current concepts and developments with special focus on molecular allergy diagnostics

Jakob, T., Rafei-Shamsabadi, D., Spillner, E., Müller, S. — Sun, 01 Jan 2017 09:29:33 +0100

Human serum substitution by artificial sera of scalable allergen reactivity based on polyclonal antibodies and chimeras of human FcγRI and IgE domains

Offermann, N., Plum, M., Hübner, U., et al. — Fri, 01 Jan 2016 09:29:33 +0100

Human sera are the first choice as controls for diagnostic applications such as immunoassays, but are limited regarding availability, varying quality, and high costs. In this study, we aimed to circumvent these limitations by the use of a chimeric adaptor molecule comprising the extracellular domains of the human FcγRI (CD64) fused with human IgE Fc domains (CD64-IgE Fc). Allergen-specific antibodies were produced in rabbits using eight different allergens, extracts, and allergen mixtures including mites, pollen, drugs, and food. Preincubation of polyclonal IgG with CD64-IgE Fc established allergen-specific artificial sera that showed comparable results for more than 20 allergens and allergen extracts in three diagnostic systems for the determination of specific IgE. The agreement for these artificial sera is within ±1 radioallergosorbent test (RAST) class. Hence, rabbit IgG complexed with the IgG-specific CD64-IgE Fc adaptor molecule could provide a substitute for human reference sera with specificity for virtually any protein of interest.

Application of recombinant antigen 5 allergens from seven allergy-relevant Hymenoptera species in diagnostics

Schiener, M., Eberlein, B., Moreno Aguilar, C., et al. — Sun, 01 Jan 2017 09:29:33 +0100

Background: Hymenoptera stings can cause severe anaphylaxis in untreated venom-allergic patients. A correct diagnosis regarding the relevant species for immunotherapy is often hampered by clinically irrelevant cross-reactivity. In vespid venom allergy, cross-reactivity between venoms of different species can be a diagnostic challenge. To address immunological IgE cross-reactivity on molecular level, seven recombinant antigens 5 of the most important Vespoidea groups were assessed by different diagnostic setups. Methods: The antigens 5 of yellow jackets, hornets, European and American paper wasps, fire ants, white-faced hornets, and Polybia wasps were recombinantly produced in insect cells, immunologically and structurally characterized, and their sIgE reactivity assessed by ImmunoCAP, ELISA, cross-inhibition, and basophil activation test (BAT) in patients with yellow jacket or Polistes venom allergy of two European geographical areas. Results: All recombinant allergens were correctly folded and structural models and patient reactivity profiles suggested the presence of conserved and unique B-cell epitopes. All antigens 5 showed extensive cross-reactivity in sIgE analyses, inhibition assays, and BAT. This cross-reactivity was more pronounced in ImmunoCAP measurements with venom extracts than in sIgE analyses with recombinant antigens 5. Dose–response curves with the allergens in BAT allowed a differentiated individual dissection of relevant sensitization. Conclusions: Due to extensive cross-reactivity in various diagnostic settings, antigens 5 are inappropriate markers for differential sIgE diagnostics in vespid venom allergy. However, the newly available antigens 5 from further vespid species and the combination of recombinant allergen-based sIgE measurements with BAT represents a practicable way to diagnose clinically relevant sensitization in vespid venom allergy.

Benefits and limitations of recombinant allergens in diagnostics of insect venom allergy

Jakob, T., Blank, S., Spillner, E. — Sun, 01 Jan 2017 09:29:33 +0100

Diagnostik der Insektengiftallergie: vom Extrakt zum Molekül

Spillner, E. — Thu, 01 Jan 2015 09:29:33 +0100

Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in T_H2 responses

Plum, M., Tjerrild, L., Raiber, T., et al. — Sun, 01 Jan 2023 09:29:33 +0100

Background: 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. Methods: 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. Results: 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. Conclusions: Our study provides evidence for the relevance of N-glycan recognition in T _H2 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.

Predominant Api m 10 sensitization as risk factor for treatment failure in honey bee venom immunotherapy

Frick, M., Fischer, J., Helbing, A., et al. — Fri, 01 Jan 2016 09:29:33 +0100

Background Component resolution recently identified distinct sensitization profiles in honey bee venom (HBV) allergy, some of which were dominated by specific IgE to Api m 3 and/or Api m 10, which have been reported to be underrepresented in therapeutic HBV preparations. Objective We performed a retrospective analysis of component-resolved sensitization profiles in HBV-allergic patients and association with treatment outcome. Methods HBV-allergic patients who had undergone controlled honey bee sting challenge after at least 6 months of HBV immunotherapy (n = 115) were included and classified as responder (n = 79) or treatment failure (n = 36) on the basis of absence or presence of systemic allergic reactions upon sting challenge. IgE reactivity to a panel of HBV allergens was analyzed in sera obtained before immunotherapy and before sting challenge. Results No differences were observed between responders and nonresponders regarding levels of IgE sensitization to Api m 1, Api m 2, Api m 3, and Api m 5. In contrast, Api m 10 specific IgE was moderately but significantly increased in nonresponders. Predominant Api m 10 sensitization (>50% of specific IgE to HBV) was the best discriminator (specificity, 95%; sensitivity, 25%) with an odds ratio of 8.444 (2.127-33.53; P = .0013) for treatment failure. Some but not all therapeutic HBV preparations displayed a lack of Api m 10, whereas Api m 1 and Api m 3 immunoreactivity was comparable to that of crude HBV. In line with this, significant Api m 10 sIgG ₄ induction was observed only in those patients who were treated with HBV in which Api m 10 was detectable. Conclusions Component-resolved sensitization profiles in HBV allergy suggest predominant IgE sensitization to Api m 10 as a risk factor for treatment failure in HBV immunotherapy.

rApi m 3 and rApi m 10 improve detection of honey bee sensitization in Hymenoptera venom-allergic patients with double sensitization to honey bee and yellow jacket venom

Frick, M., Müller, S., Bantleon, F., et al. — Thu, 01 Jan 2015 09:29:33 +0100

Structure of the omalizumab Fab

Jensen, R. K., Plum, M., Tjerrild, L., Jakob, T., Spillner, E., Andersen, G. R. — Wed, 01 Apr 2015 09:29:33 +0200

Decline of Ves v 5-specific blocking capacity in wasp venom-allergic patients after stopping allergen immunotherapy

Möbs, C., Müller, J., Rudzio, A., et al. — Sat, 07 Mar 2015 09:29:34 +0100

Complement depletion using recombinant human C3-derivatives

Sat, 01 Jan 2005 09:29:34 +0100

Method for determining an unknown PNA [peptide nucleic acid] sequence and uses thereof

Mon, 01 Jan 2007 09:29:34 +0100

Bivalent IgY antibody constructs for diagnostic and therapeutic applications

Mon, 01 Jan 2007 09:29:34 +0100

Antibody compositions specific for IgE, IgG4 and IgA epitopes as tools for the design of hypoallergenic molecules for specific immunotherapy

Thu, 01 Jan 2009 09:29:34 +0100

Modulation of effector T cell responses by local depletion of complement component C3

Tue, 01 Jan 2013 09:29:34 +0100

Controlled activation of complement components for use as endogenous adjuvant

Tue, 01 Jan 2013 09:29:34 +0100

Human IgE is efficiently produced in glycosylated and biologically active form in lepidopteran cells

Bantleon, F., Wolf, S., Seismann, H., et al. — Fri, 01 Jan 2016 09:29:34 +0100

T _H2-biased immunity to parasites and allergens is often associated with increased levels of antigen-specific and high affinity IgE. The role in reacting against minute amounts of target structures and to provoke severe anaphylactic reactions renders IgE a mechanistically outstanding isotype. IgE represents the least abundant serum antibody isotype and exhibits a variety of peculiarities including structure, extensive glycosylation and effector functions. Despite large progress in antibody technologies, however, the recombinant access to isotypes beyond IgG such as IgE still is scarce. The capacity of expression systems has to meet the complex structural conformations and the extensive posttranslational modifications that are indispensable for biological activity.In order to provide alternatives to mammalian expression systems with often low yield and a more complex glycosylation pattern we established the recombinant production of the highly complex IgE isotype in insect cells. Recombinant IgE (rIgE) was efficiently assembled and secreted into the supernatant in yields of >30 mg/L. Purification from serum free medium using different downstream processing methods provided large amounts of rIgE. This exhibited a highly specific interaction with its antigen, therapeutic anti-IgE and its high affinity receptor, the FcεRI. Lectins and glyco-proteomic analyses proved the presence of prototypic insect type N-glycans on the epsilon heavy chain. Mediator release assays demonstrated a biological activity of the rIgE comparable to IgE derived from mammalian cells. In summary the expression in insect cells provides rIgE with variant glycosylation pattern, but retained characteristics and biological activity. Therefore our data contribute to the understanding of functional and structural aspects and potential use of the IgE isotype.

Selective local inhibition of TNFR1-mediated functions at the site of antigen/allergen presentation

Wed, 25 Jun 2014 09:29:34 +0200

IgE recognition of chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology

Van Vaerenbergh, M., de Smet, L., Rafei-Shamsabadi, D., et al. — Thu, 01 Jan 2015 09:29:34 +0100

Aktueller Stand und neue Entwicklungen in der Diagnostik mit rekombinanten Insektengiftallergenen

Jakob, T., Müller, S., Rafei-Shamsabadi, D., Bantleon, F., Spillner, E. — Mon, 01 Sep 2014 09:29:34 +0200

Basophil activation test using recombinant allergens: highly specific diagnostic method complementing routine tests in wasp venom allergy

Balzer, L., Pennino, D., Blank, S., et al. — Wed, 01 Jan 2014 09:29:34 +0100

Optimierte Diagnostik der Insektengiftallergie durch rekombinante Allergene

Jakob, T., Blank, S., Spillner, E. — Thu, 01 Jan 2015 09:29:34 +0100

IgE als Zielstruktur für therapeutische Intervention

Jakob, T., Spillner, E., Lamers, M. — Fri, 01 Jan 2016 09:29:34 +0100

Recombinant allergens rarely allow identification of Hymenoptera venom-allergic patients with negative specific IgE to whole venom preparations

Fri, 01 Aug 2014 09:29:34 +0200

Publications - Immunological Biotechnology

Molekulares Design von Nanobodies als Werkzeuge in der Allergologie: Diagnostik und mehr

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

Surfaceome Profiling Suggests Potential of Anti-MUC1×EGFR Bispecific Antibody for Breast Cancer Targeted Therapy

Molecular engineering of nanobodies as tools in allergology: diagnostics and beyond

A fully human monoclonal antibody isolated from beekeepers targets the immunodominant IgE epitope of Api m 10

Extract-shaped immune repertoires as source for nanobody-based human IgE in grass pollen allergy

Construction, expression and functional characterization of an engineered antibody against tumor antigen MUC-1C

Current research and unmet needs in allergy and immunology in Germany

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

Carbohydrate Epitopes Currently Recognized as Targets for IgE Antibodies

The Honeybee Venom Major Allergen Api m 10 (Icarapin) and Its Role in Diagnostics and Treatment of Hymenoptera Venom Allergy

Structure of intact IgE and the mechanism of ligelizumab revealed by electron microscopy

The honey bee venom allergen Api m 10 displays one major IgE epitope, Api m 10160-174

Work-related allergic asthma

In Silico Evaluation of the Binding Site of Fucosyltransferase 8 and First Attempts to Synthesize an Inhibitor with Drug-Like Properties

Inhibiting phosphatase SHIP-1 enhances suboptimal IgE-mediated activation of human blood basophils but inhibits IgE-mediated activation of cultured human mast cells

Characterization of the honeybee venom proteins C1q-like protein and PVF1 and their allergenic potential

AllergoOncology: Generating a canine anti-cancer IgE against the epidermal growth factor receptor

N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Venoms of Neotropical wasps lack cross-reactive carbohydrate determinants enabling reliable protein-based specific IgE determination

Phospholipase A1-based cross-reactivity among venoms of clinically relevant Hymenoptera from Neotropical and temperate regions

Component resolved diagnostics for Hymenoptera venom allergy

AllergoOncology - the Impact of Allergy in Oncology. EAACI Position Paper.

Trapping IgE in a closed conformation by mimicking CD23 binding prevents and disrupts FcεRI interaction

Recombinant allergens as a major step in molecular allergology:

Comparing sensitivity of Hymenoptera allergen components on different diagnostic assay systems

Diagnostics in hymenoptera venom allergy: Current concepts and developments with special focus on molecular allergy diagnostics

Human serum substitution by artificial sera of scalable allergen reactivity based on polyclonal antibodies and chimeras of human FcγRI and IgE domains

Application of recombinant antigen 5 allergens from seven allergy-relevant Hymenoptera species in diagnostics

Benefits and limitations of recombinant allergens in diagnostics of insect venom allergy

Diagnostik der Insektengiftallergie: vom Extrakt zum Molekül

Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in TH2 responses

Predominant Api m 10 sensitization as risk factor for treatment failure in honey bee venom immunotherapy

rApi m 3 and rApi m 10 improve detection of honey bee sensitization in Hymenoptera venom-allergic patients with double sensitization to honey bee and yellow jacket venom

Structure of the omalizumab Fab

Decline of Ves v 5-specific blocking capacity in wasp venom-allergic patients after stopping allergen immunotherapy

Complement depletion using recombinant human C3-derivatives

Method for determining an unknown PNA [peptide nucleic acid] sequence and uses thereof

Bivalent IgY antibody constructs for diagnostic and therapeutic applications

Antibody compositions specific for IgE, IgG4 and IgA epitopes as tools for the design of hypoallergenic molecules for specific immunotherapy

Modulation of effector T cell responses by local depletion of complement component C3

Controlled activation of complement components for use as endogenous adjuvant

Human IgE is efficiently produced in glycosylated and biologically active form in lepidopteran cells

Selective local inhibition of TNFR1-mediated functions at the site of antigen/allergen presentation

IgE recognition of chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology

Aktueller Stand und neue Entwicklungen in der Diagnostik mit rekombinanten Insektengiftallergenen

Basophil activation test using recombinant allergens: highly specific diagnostic method complementing routine tests in wasp venom allergy

Optimierte Diagnostik der Insektengiftallergie durch rekombinante Allergene

IgE als Zielstruktur für therapeutische Intervention

Recombinant allergens rarely allow identification of Hymenoptera venom-allergic patients with negative specific IgE to whole venom preparations

Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in T_H2 responses