Fig. 7: FcRTAC incorporating an affinity-matured FcγRIIb binder (SK3hi) demonstrates enhanced and sustained antigen clearance efficiency.

a Alignment of SK3 and SK4 Sequences. HCDRs and LCDRs are indicated with red. b SPR-based competition assay for SK3 and SK4. NK-2-12-SK3 was immobilized on a protein A biosensor chip, followed by injection of hFcγRIIb over the flow cell. Subsequently, SK4 Fab and hFcγRIIb were injected. c Structure of the SK4 Fab in complex with FcγRIIb (PDB ID: 9M5B). The heavy and light chains of the SK4 Fab are shown in orange and light blue, respectively. The ribbon diagram of FcγRIIb is colored green, with its surface illustrated in white and the contact area with SK4 Fab highlighted in brown. d Comparison of the calculated SK3 scFv-FcγRIIb complex structure versus the SK4 Fab-FcγRIIb complex structure. e SPR-based affinity measurements of SK3 and SK3hi binding to hFcγRIIb at pH 7.4 and pH 6.0. SK3 or SK3hi was immobilized on a protein A biosensor chip, followed by injection of hFcγRIIb over the flow cell. Solid curves represent raw data, while dashed curves indicate fitted curves. f Live-cell imaging of NK-2-12-SK3hi-mediated IgE endocytosis. Cy3-labeled IgE (red) was incubated with Huvec-2b cells in the presence or absence of Cy5-labeled NK-2-12-SK3hi (white) for 2 h. Cells were stained with LysoTracker (green) and Hoechst (blue) for confocal imaging. Images were acquired using a super-resolution confocal microscope. Scale bar: 5 μm. Experiments were performed in triplicate and repeated three times with similar results. g, h The rechallenge experiment. FcγRIIb humanized mice (n = 4 mice per group) were i.p. injected with 2.5 mg/kg IgE following by injection of 2.5 mg/kg NK-2-12-SK3 or NK-2-12-SK3hi. One day later, mice were rechallenged with 2.5 mg/kg IgE without FcRTACs. Blood samples were collected to measure serum IgE levels. Data are presented as means ± SEM. Statistical comparisons were performed using Mixed-effects analysis (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, the exact P value were provided in Supplementary Table 9).