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

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.

Extract-Shaped Immune Repertoires as Source for Nanobody-Based Human IgE in Grass Pollen Allergy.

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.

Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in TH 2 responses.

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 TH 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.

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

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 epitopes 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.

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

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 coliBL21 (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.

Carbohydrate epitopes currently recognized as targets for IgE antibodies.

Until recently, glycan epitopes have not been documented by the WHO/IUIS Allergen Nomenclature Sub-Committee. This was in part due to scarce or incomplete information on these oligosaccharides, but also due to the widely held opinion that IgE to these epitopes had little or no relevance to allergic symptoms. Most IgE-binding glycans recognized up to 2008 were considered to be "classical" cross-reactive carbohydrate determinants (CCD) that occur in insects, some helminths and throughout the plant kingdom. Since 2008, the prevailing opinion on lack of clinical relevance of IgE-binding glycans has been subject to a reevaluation. This was because IgE specific for the mammalian disaccharide galactose-alpha-1,3-galactose (alpha-gal) was identified as a cause of delayed anaphylaxis to mammalian meat in the United States, an observation that has been confirmed by allergists in many parts of the world. Several experimental studies have shown that oligosaccharides with one or more terminal alpha-gal epitopes can be attached as a hapten to many different mammalian proteins or lipids. The classical CCDs also behave like haptens since they can be expressed on proteins from multiple species. This is the explanation for extensive in vitro cross-reactivity related to CCDs. Because of these developments, the Allergen Nomenclature Sub-Committee recently decided to include glycans as potentially allergenic epitopes in an adjunct section of its website (www.allergen.org). In this article, the features of the main glycan groups known to be involved in IgE recognition are revisited, and their characteristic structural, functional, and clinical features are discussed.

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

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. 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.

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.

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

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.

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.

Honeybee (Apis mellifera) venom (HBV) represents an ideal model to study the role of particular venom components in allergic reactions in sensitized individuals as well as in the eusociality of Hymenoptera species. The aim of this study was to further characterize the HBV components C1q-like protein (C1q) and PDGF/VEGF-like factor 1 (PVF1). C1q and PVF1 were produced as recombinant proteins in insect cells. Their allergenic properties were examined by determining the level of specific IgE antibodies in the sera of HBV-allergic patients (n = 26) as well as by their capacity to activate patients' basophils (n = 11). Moreover, the transcript heterogeneity of PVF1 was analyzed. It could be demonstrated that at least three PVF1 variants are present in the venom gland, which all result from alternative splicing of one transcript. Additionally, recombinant C1q and PVF1 from Spodoptera frugiperda insect cells exhibited specific IgE reactivity with approximately 38.5% of sera of HBV-allergic patients. Interestingly, both proteins were unable to activate basophils of the patients, questioning their role in the context of clinically relevant sensitization. Recombinant C1q and PVF1 can build the basis for a deeper understanding of the molecular mechanisms of Hymenoptera venoms. Moreover, the conflicting results between IgE sensitization and lack of basophil activation, might in the future contribute to the identification of factors that determine the allergenic potential of proteins.

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

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.

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

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.

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.

Diagnostics in Hymenoptera venom allergy: current concepts and developments with special focus on molecular allergy diagnostics.

BACKGROUND: The high rate of asymptomatic sensitization to Hymenoptera venom, difficulty in correctly identifying Hymenoptera and loss of sensitization over time make an accurate diagnosis of Hymenoptera venom allergy challenging. Although routine diagnostic tests encompassing skin tests and the detection of venom-specific IgE antibodies with whole venom preparations are reliable, they offer insufficient precision in the case of double sensitized patients or in those with a history of sting anaphylaxis, in whom sensitization cannot be proven or only to the presumably wrong venom. METHODS: Systematic literature research and review of current concepts of diagnostic testing in Hymenoptera venom allergy. RESULTS AND DISCUSSION: Improvements in diagnostic accuracy over recent years have mainly been due to the increasing use of molecular allergy diagnostics. Detection of specific IgE antibodies to marker and cross-reactive venom allergens improves the discrimination between genuine sensitization and cross-reactivity, and this provides a better rationale for prescribing venom immunotherapy. The basophil activation test has also increased diagnostic accuracy by reducing the number of Hymenoptera venom sensitizations overlooked with routine tests. This paper reviews current concepts of diagnostic testing in Hymenoptera venom allergy and suggests fields for further development.