The anti-FcµR CAR redirects T cells toward FcμR⁺ target cells even in the presence of soluble FcμR. (A) T cells of a healthy donor were genetically engineered with the CAR specific for FcμR and CD19, respectively. CAR expression by T cells was detected by flow cytometry using the PE-conjugated anti-IgG antibody, which binds to the CAR extracellular IgG1 domain and with the FITC-conjugated anti-CD3 antibody. Unmodified T cells (w/o CAR) were used as control. Numbers represent the percentage of T cells with CAR compared with the total number of T cells. One representative T-cell modification is shown. (B,C) T cells engineered with the anti-FcμR CAR were coincubated in increasing numbers with FcμR⁺ or FcμR⁻ 293 cells (1.5 × 10⁴ cells/well). Unmodified T cells (w/o CAR) served as control. Specific lysis of target cells was determined after 48 hours by the XTT-based viability assay (B) or after 4 hours by the calcein release assay (C). IFN-γ and IL-2 in the culture supernatants were determined by ELISA. Data represent the mean of triplicates ± SD. The assay was replicated 3 times. One representative assay is shown. (D) Specific lysis by CAR T cells was not blocked by soluble FcμR. FcµR⁺ 293 cells (1.5 × 10⁴ cells) were coincubated with anti-FcµR CAR T cells in an effector-to-tumor cell ratio of 1.5:1 in the presence of serial dilutions of FcµR-Fc protein. Specific lysis of target cells (%) was determined by the XTT-based viability assay (upper chart). Data represent the mean of triplicates ± SD. Staining of FcµR⁺ 293 cells by the anti-FcµR mAb 6B10 was not blocked by the presence of serial dilutions of FcµR-Fc (start concentration: 3.5 µg/mL) (middle chart). Data represent the MFI of triplicates ± SD. As control, binding of the 6B10 mAb to FcµR-Fc was tested (lower chart). ELISA plates were coated with an anti-human IgG antibody or an isotype-matched control antibody (each 1 µg/mL). FcµR-Fc was incubated in serial dilutions, and bound protein was recorded by the anti-FcµR mAb 6B10 followed by a biotinylated anti-rat IgG, streptavidin-peroxidase, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) as substrate. Data represent the mean of triplicates ± SD. The assay was replicated 3 times. One representative assay is shown. OD, optical density.