Antibody Conjugates Bispecific for Pollen Allergens and ICAM-1 with Potential to Prevent Epithelial Allergen Transmigration and Rhinovirus Infection.

Allergy and rhinovirus (RV) infections are major triggers for rhinitis and asthma, causing a socioeconomic burden. As RVs and allergens may act synergistically to promote airway inflammation, simultaneous treatment strategies for both causative agents would be innovative. We have previously identified the transmembrane glycoprotein intercellular adhesion molecule 1 (ICAM-1) as an anchor for antibody conjugates bispecific for ICAM-1 and Phleum pratense (Phl p) 2, a major grass pollen allergen, to block allergen transmigration through the epithelial barrier. Since ICAM-1 is a receptor for the major group RVs, we speculated that our bispecific antibody conjugates may protect against RV infection. Therefore, we created antibody conjugates bispecific for ICAM-1 and the major grass pollen allergen Phl p 5 and analyzed their capacity to affect allergen penetration and RV infection. Bispecific antibody conjugates significantly reduced the trans-epithelial migration of Phl p 5 and thus the basolateral Phl p 5 concentration and allergenic activity as determined by humanized rat basophilic leukemia cells and inhibited RV infection of cultured epithelial cells. A reduction in allergenic activity was obtained only through the prevention of allergen transmigration because the Phl p 5-specific IgG antibody did not block the allergen-IgE interaction. Our results indicate the potential of allergen/ICAM-1-specific antibody conjugates as a topical treatment strategy for allergy and RV infections.

Isolation of nanobodies with potential to reduce patients' IgE binding to Bet v 1.

BACKGROUND: Recent studies showed that a single injection of human monoclonal allergen-specific IgG antibodies significantly reduced allergic symptoms in birch pollen-allergic patients. Since the production of full monoclonal antibodies in sufficient amounts is laborious and expensive, we sought to investigate if smaller recombinant allergen-specific antibody fragments, that is, nanobodies, have similar protective potential. For this purpose, nanobodies specific for Bet v 1, the major birch pollen allergen, were generated to evaluate their efficacy to inhibit IgE-mediated responses. METHODS: A cDNA-VHH library was constructed from a camel immunized with Bet v 1 and screened for Bet v 1 binders encoding sequences by phage display. Selected nanobodies were expressed, purified, and analyzed in regards of epitope-specificity and affinity to Bet v 1. Furthermore, cross-reactivity to Bet v 1-homologues from alder, hazel and apple, and their usefulness to inhibit IgE binding and allergen-induced basophil activation were investigated. RESULTS: We isolated three nanobodies that recognize Bet v 1 with high affinity and cross-react with Aln g 1 (alder) and Cor a 1 (hazel). Their epitopes were mapped to the alpha-helix at the C-terminus of Bet v 1. All nanobodies inhibited allergic patients' polyclonal IgE binding to Bet v 1, Aln g 1, and Cor a 1 and partially suppressed Bet v 1-induced basophil activation. CONCLUSION: We identified high-affinity Bet v 1-specific nanobodies that recognize an important IgE epitope and reduce allergen-induced basophil activation revealing the first proof that allergen-specific nanobodies are useful tools for future treatment of pollen allergy.

Molecular characterization of a fungal cyclophilin allergen Rhi o 2 and elucidation of antigenic determinants responsible for IgE-cross-reactivity.

Cyclophilins are structurally conserved pan-allergens showing extensive cross-reactivity. So far, no precise information on cross-reactive IgE-epitopes of cyclophilins is available. Here, an 18-kDa IgE-reactive cyclophilin (Rhi o 2) was purified from Rhizopus oryzae, an indoor mold causing allergic sensitization. Based on LC-MS/MS-derived sequences of natural Rhi o 2, the full-length cDNA was cloned, and expressed as recombinant (r) allergen. Purified rRhi o 2 displayed IgE-reactivity and basophil degranulation with sera from all cyclophilin-positive patients. The melting curve of properly folded rRhi o 2 showed partial refolding after heat denaturation. The allergen displayed monomeric functional peptidyl-prolyl cis-trans isomerase (PPIase) activity. In IgE-inhibition assays, rRhi o 2 exhibited extensive cross-reactivity with various other cyclophilins reported as allergens from diverse sources including its homologous human autoantigen. By generating a series of deletion mutants, a conserved 69-residue (Asn81-Asn149) fragment at C terminus of Rhi o 2 was identified as crucial for IgE-recognition and cross-reactivity. Grafting of the Asn81-Asn149 fragment within the primary structure of yeast cyclophilin CPR1 by replacing its homologous sequence resulted in a hybrid molecule with structural folds similar to Rhi o 2. The IgE-reactivity and allergenic activity of the hybrid cyclophilin were greater than that of CPR1. Therefore, the Asn81-Asn149 fragment can be considered as the site of IgE recognition of Rhi o 2. Hence, Rhi o 2 serves as a candidate antigen for the molecular diagnosis of mold allergy, and determination of a major cross-reactive IgE-epitope has clinical potential for the design of next-generation immunotherapeutics against cyclophilin-induced allergies.

Past, present, and future of allergen immunotherapy vaccines.

Allergen-specific immunotherapy (AIT) is an allergen-specific form of treatment for patients suffering from immunoglobulin E (IgE)-associated allergy; the most common and important immunologically mediated hypersensitivity disease. AIT is based on the administration of the disease-causing allergen with the goal to induce a protective immunity consisting of allergen-specific blocking IgG antibodies and alterations of the cellular immune response so that the patient can tolerate allergen contact. Major advantages of AIT over all other existing treatments for allergy are that AIT induces a long-lasting protection and prevents the progression of disease to severe manifestations. AIT is cost effective because it uses the patient´s own immune system for protection and potentially can be used as a preventive treatment. However, broad application of AIT is limited by mainly technical issues such as the quality of allergen preparations and the risk of inducing side effects which results in extremely cumbersome treatment schedules reducing patient´s compliance. In this article we review progress in AIT made from its beginning and provide an overview of the state of the art, the needs for further development, and possible technical solutions available through molecular allergology. Finally, we consider visions for AIT development towards prophylactic application.

Molecular IgE sensitization profiles of urban and rural children in South Africa.

BACKGROUND: Allergens can act as disease-triggering factors in atopic dermatitis (AD) patients. The aim of the study was to elucidate the molecular IgE sensitization profile in children with and without AD living in urban and rural areas of South Africa. METHODS: Specific IgE reactivity was assessed in 166 Black South African children aged 9-38 months using a comprehensive panel of microarrayed allergens. According to clinical characterization children fell in four groups, urban AD cases (n = 32), urban controls (non-AD, n = 40), rural cases (n = 49) and rural controls (non-AD, n = 45). RESULTS: IgE reactivity to at least one of the allergens was detected in 94% of urban and 86% of rural AD children. House dust mite (HDM; 81% urban, 74% rural AD) and animal-derived allergens (50% urban, 31% rural AD) were the most frequently recognized respiratory allergens, whereas IgE to pollen allergens was almost absent. Urban AD children showed significantly higher frequency of IgE reactivity (50%) to mouse lipocalin, Mus m 1, than rural AD children (12%). The most frequently recognized food allergens were from egg (63% urban, 43% rural AD), peanut (31% vs 41%), and soybean (22% vs 27%), whereas milk sensitization was rare. α-gal-specific IgE almost exclusively occurred in rural children (AD: 14%, non-AD: 49%). CONCLUSION: Molecular allergy diagnosis detects frequent IgE sensitization to HDM, animal but not pollen allergens and to egg, peanut, and soy, but not milk allergens in African AD children. Urban AD children reacted more often to Mus m 1, whereas α-gal sensitization is more common in rural children likely due to parasite exposure.

Filling the Antibody Pipeline in Allergy: PIPE Cloning of IgE, IgG1 and IgG4 against the Major Birch Pollen Allergen Bet v 1.

Birch pollen allergy is among the most prevalent pollen allergies in Northern and Central Europe. This IgE-mediated disease can be treated with allergen immunotherapy (AIT), which typically gives rise to IgG antibodies inducing tolerance. Although the main mechanisms of allergen immunotherapy (AIT) are known, questions regarding possible Fc-mediated effects of IgG antibodies remain unanswered. This can mainly be attributed to the unavailability of appropriate tools, i.e., well-characterised recombinant antibodies (rAbs). We hereby aimed at providing human rAbs of several classes for mechanistic studies and as possible candidates for passive immunotherapy. We engineered IgE, IgG1, and IgG4 sharing the same variable region against the major birch pollen allergen Bet v 1 using Polymerase Incomplete Primer Extension (PIPE) cloning. We tested IgE functionality and IgG blocking capabilities using appropriate model cell lines. In vitro studies showed IgE engagement with FcεRI and CD23 and Bet v 1-dependent degranulation. Overall, we hereby present fully functional, human IgE, IgG1, and IgG4 sharing the same variable region against Bet v 1 and showcase possible applications in first mechanistic studies. Furthermore, our IgG antibodies might be useful candidates for passive immunotherapy of birch pollen allergy.

IgG4 drives M2a macrophages to a regulatory M2b-like phenotype: potential implication in immune tolerance.

BACKGROUND: Macrophages can be converted in vitro into immunoregulatory M2b macrophages in the presence of immune complexes (ICs), but the role of the specific subclasses IgG1 or IgG4 in this phenotypic and functional change is not known. OBJECTIVE: We aimed to refine the original method by applying precisely defined ICs of the subclasses IgG4 or IgG1 constructed by two independent methods. METHODS: Monocyte-derived macrophages (MDMs) were treated with M-CSF, followed by IL-4/IL-13 to induce the M2a allergic phenotype. To mimic unspecific or allergen-specific ICs, plates were coated with myeloma IgG1 or IgG4, or with grass pollen allergen Phl p 5 followed by recombinant human Phl p 5-specific IgG1 or IgG4. M2a polarized macrophages were then added, cultured, and examined for cellular markers and cytokines by flow cytometry, ELISA, and rtPCR. Alternatively, immune complexes with IgG1 or IgG4 were formed using protein L. RESULTS: IgG4 ICs down regulated CD163 and CD206 on M2a cells, and significantly increased IL-10, IL-6, TNFα, and CCL1 secretion, indicating a shift to an M2b-like phenotype. Treatment with IgG4 ICs resulted in expression of FcγRII and down modulation of FcγRII compared with IgG1 treated cells (P = 0.0335) or untreated cells (P < 0.00001). CONCLUSION: Immune complexes with subclasses IgG1 and IgG4 can in vitro be generated by plate absorption, and in fluid form by protein L. Cross-linking of FcγRIIb by the IgG4 subclass redirects pro-allergic M2a macrophages to an M2b-like immunosuppressive phenotype. This suggests an interplay of macrophages with IgG4 in immune tolerance, likely relevant in allergen immunotherapy.

Distinct Expression and Function of FcεRII in Human B Cells and Monocytes.

FcεRII is a multifunctional low-affinity IgER that is involved in the pathogenesis of allergic, inflammatory, and neoplastic diseases. Although discrepancies in FcεRII-mediated functions are being increasingly recognized, the consequences of FcεRII activation are not completely understood. In this study, we evaluated the expression of FcεRII on human blood cells and found that it was primarily expressed on monocytes and B cells. Although IL-4 promoted expression of the FcεRIIb isoform on B cells and monocytes, the expression of the FcεRIIa isoform was not dependent on IL-4. Furthermore, FcεRII predominantly bound allergen-IgE complexes on B cells but not on monocytes. FcεRII-mediated allergen-IgE complex uptake by B cells directed Ags to MHC class II-rich compartments. FcεRII-bearing monocytes and B cells expressed high levels of the FcεRII sheddase a disintegrin and metalloproteinase 10, which implies that they are important sources of soluble FcεRII. Moreover, we identified that IgE immune complex stimulation of FcεRII activated intracellular tyrosine phosphorylation via Syk in B cells but not in monocytes. Importantly, FcεRII-mediated signaling by allergen-IgE immune complexes increased IFN-γ production in B cells of allergic patients during the build-up phase of allergen-specific immunotherapy. Together, our results demonstrate that FcεRII mediates cell type-dependent function in allergic reactions. In addition, the results identify a novel allergen-IgE complex/FcεRII/Syk/IFN-γ pathway in allergic responses and suggest that FcεRII may play a role in regulating allergic reactions via modulating IFN-γ production in B cells.

High-density IgE recognition of the major grass pollen allergen Phl p 1 revealed with single-chain IgE antibody fragments obtained by combinatorial cloning.

The timothy grass pollen allergen Phl p 1 belongs to the group 1 of highly cross-reactive grass pollen allergens with a molecular mass of ∼25-30 kDa. Group 1 allergens are recognized by >95% of grass pollen allergic patients. We investigated the IgE recognition of Phl p 1 using allergen-specific IgE-derived single-chain variable Ab fragments (IgE-ScFvs) isolated from a combinatorial library constructed from PBMCs of a grass pollen-allergic patient. IgE-ScFvs reacted with recombinant Phl p 1 and natural group 1 grass pollen allergens. Using synthetic Phl p 1-derived peptides, the binding sites of two ScFvs were mapped to the N terminus of the allergen. In surface plasmon resonance experiments they showed comparable high-affinity binding to Phl p 1 as a complete human IgE-derived Ab recognizing the allergens' C terminus. In a set of surface plasmon resonance experiments simultaneous allergen recognition of all three binders was demonstrated. Even in the presence of the three binders, allergic patients' polyclonal IgE reacted with Phl p 1, indicating high-density IgE recognition of the Phl p 1 allergen. Our results show that multiple IgE Abs can bind with high density to Phl p 1, which may explain the high allergenic activity and sensitizing capacity of this allergen.

Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.

Allergy diagnosis based on purified allergen molecules provides detailed information regarding the individual sensitization profile of allergic patients, allows monitoring of the development of allergic disease and of the effect of therapies on the immune response to individual allergen molecules. Allergen microarrays contain a large variety of allergen molecules and thus allow the simultaneous detection of allergic patients' antibody reactivity profiles towards each of the allergen molecules with only minute amounts of serum. In this article we summarize recent progress in the field of allergen microarray technology and introduce the MeDALL allergen-chip which has been developed for the specific and sensitive monitoring of IgE and IgG reactivity profiles towards more than 170 allergen molecules in sera collected in European birth cohorts. MeDALL is a European research program in which allergen microarray technology is used for the monitoring of the development of allergic disease in childhood, to draw a geographic map of the recognition of clinically relevant allergens in different populations and to establish reactivity profiles which are associated with and predict certain disease manifestations. We describe technical advances of the MeDALL allergen-chip regarding specificity, sensitivity and its ability to deliver test results which are close to in vivo reactivity. In addition, the usefulness and numerous advantages of allergen microarrays for allergy research, refined allergy diagnosis, monitoring of disease, of the effects of therapies, for improving the prescription of specific immunotherapy and for prevention are discussed.

The human IgE repertoire.

IgE is a key mediator in allergic diseases. However, in strong contrast to other antibody isotypes, many details of the composition of the human IgE repertoire are poorly defined. The low levels of human IgE in the circulation and the rarity of IgE-producing B cells are important reasons for this lack of knowledge. In this review, we summarize the current knowledge on these repertoires both in terms of their complexity and activity, i.e. knowledge which despite the difficulties encountered when studying the molecular details of human IgE has been acquired in recent years. We also take a look at likely future developments, for instance through improvements in sequencing technology and methodology that allow the isolation of additional allergen-specific human antibodies mimicking IgE, as this certainly will support our understanding of human IgE in the context of human disease in the years to come.

Determination of allergen specificity by heavy chains in grass pollen allergen-specific IgE antibodies.

BACKGROUND: Affinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation. OBJECTIVE: We sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity. METHODS: Ten IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments. RESULTS: The shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen. CONCLUSION: Our results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens.

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

Background: Around 20% of the population in Northern and Central Europe is affected by birch pollen allergy, with the major birch pollen allergen Bet v 1 as the main elicitor of allergic reactions. Together with its cross-reactive allergens from related trees and foods, Bet v 1 causes an impaired quality of life. Hence, new treatment strategies were elaborated, demonstrating the effectiveness of blocking IgG antibodies on Bet v 1-induced IgE-mediated reactions. A recent study provided evidence for the first time that Bet v 1-specific nanobodies reduce patients´ IgE binding to Bet v 1. In order to increase the potential to outcompete IgE recognition of Bet v 1 and to foster cross-reactivity and cross-protection, we developed Bet v 1-specific nanobody trimers and evaluated their capacity to suppress polyclonal IgE binding to corresponding allergens and allergen-induced basophil degranulation. Methods: Nanobody trimers were engineered by adding isoleucine zippers, thus enabling trimeric formation. Trimers were analyzed for their cross-reactivity, binding kinetics to Bet v 1, and related allergens, and patients' IgE inhibition potential. Finally, their efficacy to prevent basophil degranulation was investigated. Results: Trimers showed enhanced recognition of cross-reactive allergens and increased efficiency to reduce IgE-allergen binding compared to nanobody monomers. Furthermore, trimers displayed slow dissociation rates from allergens and suppressed allergen-induced mediator release. Conclusion: We generated high-affine nanobody trimers that target Bet v 1 and related allergens. Trimers blocked IgE-allergen interaction by competing with IgE for allergen binding. They inhibited IgE-mediated release of biological mediators, demonstrating a promising potential to prevent allergic reactions caused by Bet v 1 and relatives.

Mapping of conformational IgE epitopes with peptide-specific monoclonal antibodies reveals simultaneous binding of different IgE antibodies to a surface patch on the major birch pollen allergen, Bet v 1.

Allergic inflammation is based on the cross-linking of mast cell and basophil-bound IgE Abs and requires at least two binding sites for IgE on allergens, which are difficult to characterize because they are often conformational in nature. We studied the IgE recognition of birch pollen allergen Bet v 1, a major allergen for >100 million allergic patients. Monoclonal and polyclonal Abs raised against Bet v 1-derived peptides were used to compete with allergic patients' IgE binding to Bet v 1 to search for sequences involved in IgE recognition. Strong inhibitions of patients' IgE binding to Bet v 1 (52-75%) were obtained with mAbs specific for two peptides comprising aa 29-58 (P2) and aa 73-103 (P6) of Bet v 1. As determined by surface plasmon resonance, mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, but only polyclonal rabbit anti-P2 and anti-P6 Abs or a combination of mAbs inhibited allergen-induced basophil degranulation. Thus, P2 and P6 define a surface patch on the Bet v 1 allergen, which allows simultaneous binding of several different IgE Abs required for efficient basophil and mast cell activation. This finding explains the high allergenic activity of the Bet v 1 allergen. The approach of using peptide-specific Abs for the mapping of conformational IgE epitopes on allergens may be generally applicable. It may allow discriminating highly allergenic from less allergenic allergen molecules and facilitate the rational design of active and passive allergen-specific immunotherapy strategies.

Passive immunization with allergen-specific antibodies.

The induction of allergen-specific IgG antibodies has been identified as a major mechanism responsible for the reduction of allergic inflammation in allergic patients treated by allergen-specific immunotherapy. Several studies suggest that allergen-specific IgG antibodies induced by vaccination with allergens block mast cell and basophil degranulation, IgE-facilitated allergen presentation to T cells and IgE production. The availability of recombinant allergens and technologies for the production of recombinant human antibodies allows engineering of allergen-specific antibodies which can be used for passive immunization (i.e., therapy) and eventually for the prevention of allergy (i.e., prophylaxis). This chapter summarizes data supporting the possible use of allergen-specific antibodies for treatment and prophylaxis. Finally, concrete approaches for the treatment and prevention of allergy based on blocking antibodies are envisioned.

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

The nasal cavity is an important site of allergen entry. Hence, it represents an organ where trans-epithelial allergen penetration and subsequent IgE-mediated allergic inflammation can potentially be inhibited. Intercellular adhesion molecule 1 (ICAM-1) is highly expressed on the surface of respiratory epithelial cells in allergic patients. It was identified as a promising target to immobilize antibody conjugates bispecific for ICAM-1 and allergens and thereby block allergen entry. We have previously characterized a nanobody specific for the major birch pollen allergen Bet v 1 and here we report the generation and characterization of ICAM-1-specific nanobodies. Nanobodies were obtained from a camel immunized with ICAM-1 and a high affinity binder was selected after phage display (Nb44). Nb44 was expressed as recombinant protein containing HA- and His-tags in Escherichia coli (E.coli) and purified via affinity chromatography. SDS-PAGE and Western blot revealed a single band at approximately 20 kDa. Nb44 bound to recombinant ICAM-1 in ELISA, and to ICAM-1 expressed on the human bronchial epithelial cell line 16HBE14o- as determined by flow cytometry. Experiments conducted at 4°C and at 37°C, to mimic physiological conditions, yielded similar percentages (97.2 ± 1.2% and 96.7 ± 1.5% out of total live cells). To confirm and visualize binding, we performed immunofluorescence microscopy. While Texas Red Dextran was rapidly internalized Nb44 remained localized on the cell surface. Additionally, we determined the strength of Nb44 and ICAM-1 interaction using surface plasmon resonance (SPR). Nb44 bound ICAM-1 with high affinity (10-10 M) and had slow off-rates (10-4 s-1). In conclusion, our results showed that the selected ICAM-1-specific nanobody bound ICAM-1 with high affinity and was not internalized. Thus, it could be further used to engineer heterodimers with allergen-specific nanobodies in order to develop topical treatments of pollen allergy.

High-affinity IgE recognition of a conformational epitope of the major respiratory allergen Phl p 2 as revealed by X-ray crystallography.

We report the three-dimensional structure of the complex between the major respiratory grass pollen allergen Phl p 2 and its specific human IgE-derived Fab. The Phl p 2-specific human IgE Fab has been isolated from a combinatorial library constructed from lymphocytes of a pollen allergic patient. When the variable domains of the IgE Fab were grafted onto human IgG1, the resulting Ab (huMab2) inhibited strongly the binding of allergic patients' IgE to Phl p 2 as well as allergen-induced basophil degranulation. Analysis of the binding of the allergen to the Ab by surface plasmon resonance yielded a very low dissociation constant (K(D) = 1.1 x 10(-10) M), which is similar to that between IgE and Fcepsilon;RI. The structure of the Phl p 2/IgE Fab complex was determined by x-ray crystallography to 1.9 A resolution revealing a conformational epitope (876 A(2)) comprised of the planar surface of the four-stranded anti-parallel beta-sheet of Phl p 2. The IgE-defined dominant epitope is discontinuous and formed by 21 residues located mostly within the beta strands. Of the 21 residues, 9 interact directly with 5 of the 6 CDRs (L1, L3, H1, H2, H3) of the IgE Fab predominantly by hydrogen bonding and van der Waals interactions. Our results indicate that IgE Abs recognize conformational epitopes with high affinity and provide a structural basis for the highly efficient effector cell activation by allergen/IgE immune complexes.