Abstract
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.
Highlights
Birch pollen allergy is among the most common allergies in Central and Northern Europe, with a prevalence of up to 16% [1]
The molecular key player in any allergy is IgE, which interacts with IgE receptors FcεRI and CD23 on the surface of effector cells [6]
Binding to the high-affinity receptor FcεRI on mast cells and basophils is of major importance for the immediate allergic reaction: Upon allergen binding to IgE, FcεRs are cross-linked and cells release mediators that are responsible for typical allergy symptoms [7]
Summary
Birch pollen allergy is among the most common allergies in Central and Northern Europe, with a prevalence of up to 16% [1]. We applied PIPE cloning with the objective of deriving IgG1 and IgG4 isotypes from a known IgE antibody against the major birch pollen allergen Bet v 1 [27] and elucidating their target antigen engagement. We determined the capab5iloitfi2e2s of our IgG antibodies to block IgE binding to Bet v 1 in these allergic donor samples (see Figure 3b for a scheme describing the method). Wellesfwurethreeremitohreerapdrdee-dtreeiathteedr with M041B8etIgvE1oarloanne iosrotByept ev c1oinntcrooml tboindaettieornmwinithe ianntearnntia-IlgisEatainotnibsopdeyc,itfioceitsyt.abWlieshfuarpthoseirtmiveorceonatdrodlefdoreither Bet vB1etavlo1nienoterrnBaeltisvat1ioinnvcioamarbtiifnicaitailornecwepitthoracnroasnsltiin-IkginEga. ntibody, to establish a positive control for Bet v 1 internalisation via artificial receptor crosslinking Both of the cell lines expressed CD23, but not FcεRI, as confirmed by flow cytometry (Figure S5). Data are represented as means ± SD of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001
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