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.