Kerstin Greunke

Dissecting cross-reactivity in hymenoptera venom allergy by circumvention of α-1,3-core fucosylation.

Hymenoptera venom allergy is known to cause life-threatening and sometimes fatal IgE-mediated anaphylactic reactions in allergic individuals. About 30-50% of patients with insect venom allergy have IgE antibodies that react with both honeybee and yellow jacket venom. Apart from true double sensitisation, IgE against cross-reactive carbohydrate determinants (CCD) are the most frequent cause of multiple reactivities severely hampering the diagnosis and design of therapeutic strategies by clinically irrelevant test results. In this study we addressed allergenic cross-reactivity using a recombinant approach by employing cell lines with variant capacities of alpha-1,3-core fucosylation. The venom hyaluronidases, supposed major allergens implicated in cross-reactivity phenomena, from honeybee (Api m 2) and yellow jacket (Ves v 2a and its putative isoform Ves v 2b) as well as the human alpha-2HS-glycoprotein as control, were produced in different insect cell lines. In stark contrast to production in Trichoplusia ni (HighFive) cells, alpha-1,3-core fucosylation was absent or immunologically negligible after production in Spodoptera frugiperda (Sf9) cells. Consistently, co-expression of honeybee alpha-1,3-fucosyltransferase in Sf9 cells resulted in the reconstitution of CCD reactivity. Re-evaluation of differentially fucosylated hyaluronidases by screening of individual venom-sensitised sera emphasised the allergenic relevance of Api m 2 beyond its carbohydrate epitopes. In contrast, the vespid hyaluronidases, for which a predominance of Ves v 2b could be shown, exhibited pronounced and primary carbohydrate reactivity rendering their relevance in the context of allergy questionable. These findings show that the use of recombinant molecules devoid of CCDs represents a novel strategy with major implications for diagnostic and therapeutic approaches.

Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy.

Abstract The generation and use of avian antibodies is of increasing interest in a wide variety of applications within the life sciences. Due to their phylogenetic distance, mechanisms of immune diversification and the way in which they deposit IgY immunoglobulin in the egg yolk, chickens provide a number of advantages compared to mammals as hosts for immunization. These advantages include: the one-step purification of antibodies from egg yolk in large amounts facilitates having a virtually continuous supply; the epitope spectrum of avian antibodies potentially grants access to novel specificities; the broad absence of cross-reactivity with mammalian epitopes avoids assay interference and improves the performance of immunological techniques. The polyclonal nature of IgY antibodies has limited their use since avian hybridoma techniques are not well established. Recombinant IgY, however, can be generated from mammalian monoclonal antibodies which makes it possible to further exploit the advantageous properties of the IgY scaffold. Moreover, cloning and selecting the immune repertoire from avian organisms is highly efficient, yielding antigen-specific antibody fragments. The recombinant approach is well suited to circumvent any limitations of polyclonal antibodies. This review presents comprehensive information on the generation, purification, modification and applications of polyclonal and monoclonal IgY antibodies.

Evaluation of different glycoforms of honeybee venom major allergen phospholipase A2 (Api m 1) produced in insect cells.

Allergic reactions to hymenoptera stings are one of the major reasons for IgE-mediated anaphylaxis. However, proper diagnosis using venom extracts is severely affected by molecular cross-reactivity. In this study recombinant honeybee venom major allergen phospholipase A2 (Api m 1) was produced for the first time in insect cells. Using baculovirus infection of different insect cell lines allergen versions providing a varying degree of cross-reactive carbohydrate determinants as well as a non glycosylated variant could be obtained as secreted soluble proteins in high yields. The resulting molecules were analyzed for their glycosylation and proved to show advantageous properties regarding cross-reactivity in sIgE-based assays. Additionally, in contrast to the enzymatically active native protein the inactivated allergen did not induce IgE-independent effector cell activation. Thus, insect cell-derived recombinant Api m 1 with defined CCD phenotypes might provide further insights into hymenoptera venom IgE reactivities and contribute to an improved diagnosis of hymenoptera venom allergy.

Close-up of the Immunogenic α1,3-Galactose Epitope as Defined by a Monoclonal Chimeric Immunoglobulin E and Human Serum Using Saturation Transfer Difference (STD) NMR

Background: Antibody binding to xenobiotic α-Gal structures mediates anaphylaxis. Results: Humanized IgE antibodies exhibit recognition footprints similar to serum immunoglobulins but have no capability of effector cell activation. Conclusion: Recognition of the α-Gal epitope is based on the terminal disaccharide, but interaction does not imply cellular activation. Significance: These data provide the first insights into the recognition of carbohydrate IgE epitopes. Anaphylaxis mediated by carbohydrate structures is a controversially discussed phenomenon. Nevertheless, IgE with specificity for the xenotransplantation antigen α1,3-Gal (α-Gal) are associated with a delayed type of anaphylaxis, providing evidence for the clinical relevance of carbohydrate epitopes in allergy. The aim of this study was to dissect immunoreactivity, interaction, and fine epitope of α-Gal-specific antibodies to obtain insights into the recognition of carbohydrate epitopes by IgE antibodies and their consequences on a molecular and cellular level. The antigen binding moiety of an α-Gal-specific murine IgM antibody was employed to construct chimeric IgE and IgG antibodies. Reactivity and specificity of the resulting antibodies were assessed by means of ELISA and receptor binding studies. Using defined carbohydrates, interaction of the IgE and human serum was assessed by mediator release assays, surface plasmon resonance (SPR), and saturation transfer difference NMR analyses. The α-Gal-specific chimeric IgE and IgG antibodies were proven functional regarding interaction with antigen and Fc receptors. SPR measurements demonstrated affinities in the micromolar range. In contrast to a reference antibody, anti-Gal IgE did not induce mediator release, potentially reflecting the delayed type of anaphylaxis. The α1,3-Gal epitope fine structures of both the recombinant IgE and affinity-purified serum were defined by saturation transfer difference NMR, revealing similar contributions of carbohydrate residues and participation of both galactose residues in interaction. The antibodies generated here constitute the principle underlying α1,3-Gal-mediated anaphylaxis. The complementary data of affinity and fine specificity may help to elucidate the recognition of carbohydrates by the adaptive immune response and the molecular requirements of carbohydrate-based anaphylaxis.

Comparative expression of different antibody formats in mammalian cells and Pichia pastoris.

Generation of recombinant antibody fragments has been advanced by phage display technology but their broad use in biochemical or analytical applications is often hindered by their univalence. For enhancement of functional affinity and overall applicability, the fusion of scFvs (single‐chain variable fragments) to IgG constant domains has become an attractive approach. In order to evaluate characteristics and expression behaviour of different IgG‐analogous antibody formats, we fused an scFv to different portions of the heavy chain constant region of human IgG1. Two types of antibodies, an scFv–CH2–3 antibody and an scFv–CH1–3 antibody, a new intermediate with retained CH1 domain, were generated with or without an affinity tag for purification. Additionally, the scFv was reconverted into the heterotetrameric IgG molecule. To allow a reliable comparison of expression behaviours of different antibodies, we established vector systems that allow isogenetic and efficient expression of the recombinant antibodies based on site‐specific recombination. Upon recombinant expression in mammalian cells and the methylotrophic yeast Pichia pastoris, disulfide‐linked and glycosylated oligomers were obtained. Establishment of isogenetic cell lines revealed that the presence of the CH1 domain is not critical for secretion efficiency. Reactivity of the different constructs with antigen and Fc receptors was verified by ELISA, surface plasmon resonance approaches, as well as FACS analysis of HEK‐293 cells (human embryonic kidney cells) stably transfected with human FcγRI (high‐affinity IgG receptor) (CD64). In summary, the results obtained provide evidence for comparable behaviour of the different antibody formats and the vectors for isogenetic expression will contribute to a broader application of phage display‐derived antibodies.

Recombinant IgY for improvement of immunoglobulin-based analytical applications

OBJECTIVES In order to provide superior tools for diagnostic approaches and to prevent assay interference and background binding, the objective of this study was the establishment and evaluation of monoclonal IgY which are phylogenetically distant from mammalian immunoglobulins but have been unavailable so far. DESIGN AND METHODS Human, murine and avian monoclonal model antibodies were established and produced in mammalian cells. Their interaction with human serum components and Fc gamma receptors was compared by ELISA and fluorescence activated cell sorting (FACS). RESULTS The use of monoclonal IgY in contrast to mammalian antibodies prevented interference phenomena in absorbance measurements generated by human sera containing rheumatoid factor (RF) or heterophilic antibodies. Additionally, monoclonal IgY exhibited no interaction with the human and murine high-affinity receptor FCGR1 (CD64) and human low affinity receptor FCGR3a (CD16A). CONCLUSIONS The data obtained demonstrate the advantageous behaviour of monoclonal IgY as detection or capture antibodies compared to conventional mammalian immunoglobulins and provide a strategy for improvement of assay performance and accuracy.

Establishment of hapten-specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries

Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6‐trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single‐chain antibody fragments) specific for the TNT‐surrogate TNP (2,4,6‐trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP‐specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP‐specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay‐based techniques.

Quantitation of serum IgE by using chimeras of human IgE receptor and avian immunoglobulin domains

Anti-IgE therapeutics represent an efficient approach in the management of IgE-mediated allergic asthma. However, monitoring the reduction of IgE levels into a therapeutically efficient range requires the determination of residual serum IgE. We established an analytical approach to distinguish free and anti-IgE complexed serum IgE based on soluble derivatives of the human high-affinity IgE receptor. Soluble receptor derivatives represent an ideal means to analyze receptor antagonism by any ligand or blocking antibody. Therefore, the FcεRI ectodomain was fused with avian IgY constant domains that circumvent susceptibility to interference phenomena and improve assay performance. After production in HEK293 cells, subsequent characterization by enzyme-linked immunosorbent assay and immunoblotting confirmed the suitability of avian IgY constant domains for immobilization and detection purposes. To provide further insights into the different IgE reactivities, free allergen-specific IgE was also determined. Monitoring of sera from omalizumab-treated patients during the course of therapy revealed the applicability for assessment of omalizumab-complexed versus noncomplexed serum IgE. These parameters may allow correlation to clinical responses during anti-IgE therapy with the perspective of biomonitoring.