3.4 IgG1 and IgG4 bind to human FcγRIIβ with similar affinity
In comparison to other Fcγ receptors, the affinity of IgG antibodies to the FcγRIIβ is low even if immune-complexed to antigens. We nevertheless measured the affinity of IgG1 and IgG4 antibodies to FcγRIIb (CD32b) by Biolayer Interferometry using Octet technology 42. To this end, biotinylated recombinant human FcγRIIβ was immobilized on streptavidin biosensor before incubation with either F127, G078 and A044 in either IgG1 or IgG4 format. As shown in Figure 4, IgG1 and IgG4 antibodies bound with similar affinity to recombinant FcγRIIβ (Figure 4A and Table 1).
We also investigated the effects of immune complex formation on the binding of IgG1 or IgG4 to FcγRIIb by BLI, performing the association step with IgG1 and IgG4 F127 either in monomeric or in complex form with dimeric Fel d 1 (Figure 4 C). The results showed similar association rate (kon) for monomeric and complexed IgG1 and IgG4 whereas the dissociation rate (koff) was 10 times less for the uncomplexed forms of IgG1 and IgG4 indicating an overall higher binding for IgG immune complexes than for IgGs alone. Hence, antibody subclass may have a limited influence on FcγRIIβ binding in both monomeric and complexed form thereby confirming the results obtained in the above cellular assays.