In vitro binding activity of 2.4G2 scFv-MSA for murine FcγRIII/IIB and in vivo pharmacokinetics. (A) RAW264.7 macrophage-like cell line, known to express FcγRIII and FcγRIIB, was stained with 0.11 µM 2.4G2 scFv-MSA (10 µg/mL) in the presence of vehicle control (PBS) or an equimolar amount of 2.4G2 or HSA (as competitive inhibitors). Residual bound 2.4G2 scFv-MSA was detected by anti-His-PE. Data representative of 4 independent experiments. (B) To analyze the ability of 2.4G2 scFv-MSA to inhibit PE-labeled 2.4G2 binding, RAW264.7 cells were stained with 0.013 µM (2 µg/mL) PE-labeled 2.4G2 in the presence of 0.11 µM (10 µg/mL) 2.4G2 scFv-MSA, HSA, 2.4G2, or PBS. Data representative of 5 independent experiments. (C) For in vivo pharmacokinetic analysis, mice were injected with 80 µg 2.4G2 scFv-MSA or ∼200 µg 2.4G2 Fab, and then bled after 0.5, 2, 4, 8, 24, and 48 hours. Serum samples were prepared and used to stain RAW264.7 cells; bound 2.4G2 scFv-MSA was detected by anti-His-PE, and bound 2.4G2 Fab in serum was detected by anti-rat IgG-κ chain-PE. The level of remaining serum protein was expressed as a percentage of mean log intensity at 0.5 hour. n = 6–8, from 3 independent experiments. Data are presented as mean ± standard error of the mean.