High-Density IgE Recognition of the Major Grass Pollen Allergen Phl p 1 Revealed with Single-Chain IgE Antibody Fragments Obtained by Combinatorial Cloning

Abstract

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.

Allergome-wide peptide microarrays enable epitope deconvolution in allergen-specific immunotherapy

“…Past studies have demonstrated the extent of crossreactivity of a few human mAbs to sequence variants of allergens and allergen protein families, exemplified by studies of epitopes on Bet v 1 and Phl p 1. [22][23][24][25][26] We now greatly extend these findings and define numerous epitopes on Bet v 1, some of which are likely structurally overlapping. Serum antibodies also recognize isoallergens, isoforms, and cross-reacting allergens.…”

“…In the case of the beta-expansin-specific immune response, we demonstrated that the majority of IgG that bind linear epitopes target the N-terminal domain of the allergen (Fig 3; Figs E3 and E13). Although specific antibodies that bind this domain have been isolated, 24 this finding contrasts past studies that have defined the C-terminal domain to be the major target of allergen-specific IgE. 26 Further studies are thus required to define whether AIT, as performed using the Alutard vaccine, primarily induce humoral immunity to a domain not primarily targeted by IgE, or whether this domain is just more prone than the C-terminal domain to induce antibodies targeting linear epitopes.…”

Isolation of a high‐affinity Bet v 1‐specific IgG‐derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments

“…Phagemid DNAs were transformed into the nonsuppressor E. coli strain HB2151 for expression of soluble ScFvs carrying a peptide E tag at the C‐terminus . Additionally, ScFvs carrying a C‐terminal His tag were constructed as described . Soluble Bet v 1‐specific ScFvs were purified by affinity chromatography .…”

Nanobodies—Useful Tools for Allergy Treatment?

“…native antibodies with the preservation of the natural VH and VL pairing including hybridoma technology, Epstein-Barr-Virus (EBV) transformation, single B cell sorting and cloning and HumAb mice (transgenic mice that produce fully human antibodies) (18,(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49). In parallel, versatile approaches were developed to generate non-genuine antibodies by random combination of VH and VL chains, i.e., combinatorial Fab/ScFv libraries or (semi-) synthetic libraries (37,38,(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60). Based on PCR amplification as strong tool to depict large antibody repertoires and phage display to screen these large repertoires, many recombinant allergen-specific antibody fragments (Fabs or ScFvs) were isolated (37,38,(50)(51)(52)(53)(54)(55)(56)(58)(59)(60)(61)(62)(63)(64).…”