FIGURE LEGENDS
Figure 1. Binding and protective role of allergen-specific IgG antibodies in mast cell degranulation. A) Mice were immunized with the vaccine Fel d 1 coupled to Qβ-VLP. By mammalian cell display, three monoclonal antibodies (G078, A044, F127) were cloned in IgG and IgE formats that recognize non-overlapping B cell epitopes. B) Anti-Fel d 1 IgG antibody (e.g. F127) having the same epitope specificity as anti-Fel d 1 IgE (e.g. F127) neutralizes Fel d 1. C) Anti-Fel d 1 IgG antibody (e.g. F127) having a different specificity as anti-Fel d 1 IgE (e.g. G078) inhibit degranulation via FcγRIIb.
Figure 2. IgG 1 and IgG4 mAbs equally well prevent basophil activation from cat allergic subjects. Blood from two (A and B) cat allergic subjects were collected and incubated with the amount of recombinant Fel d 1 (subject A: 0.015 nM; subject B: 0.022 nM) that causes ∼50% of their basophils to degranulate. That respective amount of Fel d 1 was also pre-mixed with 3, 30, and 300 nM of Fel d 1 specific mAbs either in the format of IgG1 or IgG4. The percentage of CD63+ within the CCR3+ basophil population was measured.
Figure 3. IgG1 and IgG4 prevent degranulation by neutralization and signalling via the inhibitory receptor FcγRIIb. Blood from two non-allergic subjects was collected and primed with Fel d 1 specific IgE. A) Basophils were primed either with IgE A044, F127 or G078. Primed basophils were treated with recombinant Fel d 1 alone or pre-mixed with the same epitope expressed as IgG1 or IgG4. B) Basophils were primed either with IgE A044, F127 or G078. Primed basophils were treated with recombinant Fel d 1 alone or pre-mixed with the IgG1 or IgG4 antibodies exhibiting the other two epitopes than the one used for priming. C) Basophils were primed either with IgE A044, F127 or G078. Primed basophils were treated with recombinant Fel d 1 alone or pre-mixed with a combination of two IgG1 or IgG4 antibodies exhibiting the other two epitopes than the one used for priming. D) % inhibition of basophil activation when basophils were primed either with IgE A044, F127 or G078 and treated with Fel d 1 pre-mixed with either IgG1 or IgG4 antibodies.
Figure 4. BLI sensorgrams illustrating the interaction IgG1 and IgG4 antibodies to FcγRIIb. A) Binding of monomeric F127, G078 and A044 in either IgG1 or IgG4 format. B) Binding of IgG1 and IgG4 F127 either as monomer or in pre-complexed form with Fel d 1. The on-rate (kon) and off-rate (koff) constants were compared. In both assays association and dissociation were performed 300s.