Fig. 3.

Different affinities of anti-MAGE antibodies to FcγR: a Influence of IgG isotype on receptor affinity: Binding of murine antibody-opsonized MAGE-A3 protein to different Fcγ receptors (FcγR) expressed on THP-1 cell-line and primary NK cells, respectively, measured by flow cytometry. Negative control was loaded MAGE-A3 protein. Antibody concentrations: 0.2 μg/ml (dotted), 2 μg/ml (dashed), 20 μg/ml (solid). b Antigen binding was shown by intracellular staining of serial antibody dilutions of either normal (gly) or deglycosylated (de-gly) antibodies in MAGE-A3 expressing U266 cells, measured by flow cytometry. MFI values of one representative out of 3 independent experiments. Curves describe non-linear one site specific binding. c Anti-MAGE antibodies 6C1 and 57B were deglycosylated using PNGase F, cleaving Fc residue Asn297. Original antibody (gly); deglycosylated antibody (de-gly). One representative out of three experiments is shown. Antibody concentrations: 0.2 μg/ml (dotted), 2 μg/ml (dashed), 20 μg/ml (solid)