In vitro characterization of CR2Fc. CR2Fc binds to C3-opsonized cells and increases Fc opsonization by amplifying its own ligand. (A) CR2Fc binding: EL4 cells sensitized with anti-GD2 14G2a mAbs were incubated with NMS and 20 μg/mL AlexaFluor-488–labeled CR2Fc added during serum incubation or after serum incubation (after washing). CR2Fc binding in the absence of mAb sensitization (CR2Fc only) was also determined. (B) CR2Fc-enhanced complement activation: EL4 cells were incubated in 5% or 50% NMS, with 14G2a and CR2Fc as indicated, and C3a levels in supernatant determined by Western blot and densitometry. (C) CR2Fc-enhanced complement-dependent EL4 lysis: EL4 cells were incubated in 5% or 50% normal rat serum, and with 14G2a and CR2Fc as indicated. (D) CR2Fc-enhanced complement-dependent B16 lysis: B16 cells were incubated in 50% normal rat serum, and with anti-gp75 TA99 mAb and CR2Fc as indicated. Cell lysis determined by measuring lactate dehydrogenase release. Data are mean ± SEM; n = 3.