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.

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.

Periostin in Allergy and Inflammation.

Matricellular proteins are involved in the crosstalk between cells and their environment and thus play an important role in allergic and inflammatory reactions. Periostin, a matricellular protein, has several documented and multi-faceted roles in health and disease. It is differentially expressed, usually upregulated, in allergic conditions, a variety of inflammatory diseases as well as in cancer and contributes to the development and progression of these diseases. Periostin has also been shown to influence tissue remodelling, fibrosis, regeneration and repair. In allergic reactions periostin is involved in type 2 immunity and can be induced by IL-4 and IL-13 in bronchial cells. A variety of different allergic diseases, among them bronchial asthma and atopic dermatitis (AD), have been shown to be connected to periostin expression. Periostin is commonly expressed in fibroblasts and acts on epithelial cells as well as fibroblasts involving integrin and NF-κB signalling. Also direct signalling between periostin and immune cells has been reported. The deposition of periostin in inflamed, often fibrotic, tissues is further fuelling the inflammatory process. There is increasing evidence that periostin is also expressed by epithelial cells in several of the above-mentioned conditions as well as in cancer. Augmented periostin expression has also been associated with chronic inflammation such as in inflammatory bowel disease (IBD). Periostin can be expressed in a variety of different isoforms, whose functions have not been elucidated yet. This review will discuss potential functions of periostin and its different isoforms in allergy and inflammation.

The H2B ubiquitin-protein ligase RNF40 is required for somatic cell reprogramming.

Direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) requires a resetting of the epigenome in order to facilitate a cell fate transition. Previous studies have shown that epigenetic modifying enzymes play a central role in controlling induced pluripotency and the generation of iPSC. Here we show that RNF40, a histone H2B lysine 120 E3 ubiquitin-protein ligase, is specifically required for early reprogramming during induced pluripotency. Loss of RNF40-mediated H2B monoubiquitination (H2Bub1) impaired early gene activation in reprogramming. We further show that RNF40 contributes to tissue-specific gene suppression via indirect effects by controlling the expression of the polycomb repressive complex-2 histone methyltransferase component EZH2, as well as through more direct effects by promoting the resolution of H3K4me3/H3K27me3 bivalency on H2Bub1-occupied pluripotency genes. Thus, we identify RNF40 as a central epigenetic mediator of cell state transition with distinct functions in resetting somatic cell state to pluripotency.

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.

Schwann cells, but not Oligodendrocytes, Depend Strictly on Dynamin 2 Function.

Myelination requires extensive plasma membrane rearrangements, implying that molecules controlling membrane dynamics play prominent roles. The large GTPase dynamin 2 (DNM2) is a well-known regulator of membrane remodeling, membrane fission, and vesicular trafficking. Here, we genetically ablated Dnm2 in Schwann cells (SCs) and in oligodendrocytes of mice. Dnm2 deletion in developing SCs resulted in severely impaired axonal sorting and myelination onset. Induced Dnm2 deletion in adult SCs caused a rapidly-developing peripheral neuropathy with abundant demyelination. In both experimental settings, mutant SCs underwent prominent cell death, at least partially due to cytokinesis failure. Strikingly, when Dnm2 was deleted in adult SCs, non-recombined SCs still expressing DNM2 were able to remyelinate fast and efficiently, accompanied by neuropathy remission. These findings reveal a remarkable self-healing capability of peripheral nerves that are affected by SC loss. In the central nervous system, however, we found no major defects upon Dnm2 deletion in oligodendrocytes.

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.

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

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 sIgG4 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.

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

TH2-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.

Promotion of periostin expression contributes to the migration of Schwann cells.

Neuregulin ligands and their ErbB receptors are important for the development of Schwann cells, the glial cells of the peripheral nervous system (PNS). ErbB3 deficiency is characterized by a complete loss of Schwann cells along axons of the peripheral nerves, impaired fasciculation and neuronal cell death. We performed comparative gene expression analysis of dorsal root ganglia (DRG) explant cultures from ErbB3-deficient and wild-type mice in order to identify genes that are involved in Schwann cell development and migration. The extracellular matrix (ECM) gene periostin was found to exhibit the most prominent down regulation in ErbB3-deficient DRG. Expression analysis revealed that the periostin-expressing cell population in the PNS corresponds to Schwann cell precursors and Schwann cells, and is particularly high in migratory Schwann cells. Furthermore, stimulation of Schwann cells with neuregulin-1 (NRG1) or transforming growth factor ß (TGFß-1) resulted in an upregulation of periostin expression. Interestingly, DRG explant cultures of periostin-deficient mice revealed a significant reduction of the number of migrating Schwann cells. These data demonstrate that the expression of periostin is stimulated by ErbB ligand NRG1 and influences the migration of Schwann cell precursors.

Recombinant IgE antibody engineering to target EGFR.

Monoclonal antibodies have become a mainstay for the targeted treatment of cancer today. Some of the most successful targets of monoclonal antibodies are constituted by the epidermal growth factor receptor family spearheaded by the epidermal growth factor receptor (EGFR). Prompted by studies indicating that IgE compared to IgG may harness alternate effector functions to eradicate malignant cells, we addressed the establishment, engineering, and the potential tumoricidal effects of recombinant anti-EGFR IgE. Therefore, two different therapeutic EGFR-specific antibodies, 225 and 425, were chosen for re-cloning into different chimeric IgE and IgG formats and produced in human cells. Simultaneous antibody binding to the sEGFR demonstrated accessibility of both epitopes for recombinant IgE. Proliferation and cytotoxicity assays demonstrated signal blocking and effector mediating capability of IgE isotypes. Pronounced degranulation in the presence of sEGFR upon activation exclusively with two IgE antibodies verified the epitope proximity and provides evidence that tumor-targeting by anti-EGFR IgE is safe with regard to soluble target structures. Degranulation mediated by tumor cells expressing EGFR could be demonstrated for singular and combined IgE antibodies; however, use of two IgE specificities was not superior to use of one IgE alone. The data suggest that the surface distribution of EGFR is optimally suited to mount a robust effector cell trigger and corroborate the potential and specificity of the IgE/IgE receptor network to react to xenobiotic or pathogenic patterns for targeting malignancies.

Dynamin 2 mutations in Charcot-Marie-Tooth neuropathy highlight the importance of clathrin-mediated endocytosis in myelination.

Mutations in dynamin 2 (DNM2) lead to dominant intermediate Charcot-Marie-Tooth neuropathy type B, while a different set of DNM2 mutations cause autosomal dominant centronuclear myopathy. In this study, we aimed to elucidate the disease mechanisms in dominant intermediate Charcot-Marie-Tooth neuropathy type B and to find explanations for the tissue-specific defects that are associated with different DNM2 mutations in dominant intermediate Charcot-Marie-Tooth neuropathy type B versus autosomal dominant centronuclear myopathy. We used tissue derived from Dnm2-deficient mice to establish an appropriate peripheral nerve model and found that dominant intermediate Charcot-Marie-Tooth neuropathy type B-associated dynamin 2 mutants, but not autosomal dominant centronuclear myopathy mutants, impaired myelination. In contrast to autosomal dominant centronuclear myopathy mutants, Schwann cells and neurons from the peripheral nervous system expressing dominant intermediate Charcot-Marie-Tooth neuropathy mutants showed defects in clathrin-mediated endocytosis. We demonstrate that, as a consequence, protein surface levels are altered in Schwann cells. Furthermore, we discovered that myelination is strictly dependent on Dnm2 and clathrin-mediated endocytosis function. Thus, we propose that altered endocytosis is a major contributing factor to the disease mechanisms in dominant intermediate Charcot-Marie-Tooth neuropathy type B.