The anti-FcμR scFv-Fc antibody conserves the binding specificity of the parental 6B10 antibody. (A) The anti-FcµR scFv-Fc antibody, which represents the extracellular CAR domain, specifically binds to FcµR⁺ cells. FcµR⁺ 293 cells (2 × 10⁵ cells) were incubated in serial dilutions starting from 1000 ng/mL with anti-FcµR scFv-Fc. For control, 293 FcµR⁻ cells were incubated with the anti-FcµR scFv-Fc protein (1000 ng/mL). Bound protein was detected by flow cytometry using the PE-conjugated anti-IgG antibody. Data represent the MFI of triplicates ± standard deviation (SD). (B) The anti-FcµR scFv-Fc binds specifically to Mec-1 and primary CLL cells. Mec-1 cells and patient’s CLL cells were incubated with the anti-FcµR scFv-Fc or, for control, with the anti-CEA scFv-Fc antibody of irrelevant specificity (each 1000 ng/mL). Bound scFv-Fc was detected by flow cytometry, and histograms were overlayed (light gray: anti-CEA scFv-Fc; dark gray: anti-FcµR scFv-Fc). (C) The anti-FcµR scFv-Fc retains binding specificity of the parental 6B10 mAb. CLL cells (n = 6) and B and T cells of healthy blood donors (n = 3 each) were incubated with anti-FcµR scFv-Fc or, for control, with the anti-CEA scFv-Fc (each 1000 ng/mL) and detected by the PE-conjugated anti-human IgG antibody (1:500). T cells and B cells were identified by anti-CD3 and anti-CD19 mAbs, and the MFI of bound scFv-Fc proteins was determined. Data represent mean values ± SD of the various donors. Statistical analysis was performed using the Student t test (**P < .01; ns, not significant).