Anti-FcμR CAR T cells specifically lyse CLL cells but spare healthy B cells. (A) Mec-1 cells express FcμR and CD19 as revealed by flow cytometry after staining with the FITC-conjugated anti-FcμR mAb 6B10 or the PE-conjugated anti-CD19 antibody (bold line). Staining with an isotype-matched antibody served as control (gray histograms). T cells engineered with the anti-FcμR CAR and the anti-CD19 CAR, respectively, were coincubated in increasing numbers with FcμR⁺ CD19⁺ Mec-1 leukemia cells (1.5 × 10⁴ cells/well). Unmodified T cells (w/o CAR) served as control. Specific lysis toward Mec-1 cells was determined after 48 hours by the XTT-based viability assay. (B) T cells from healthy donors were engineered with the anti-FcμR CAR and anti-CD19 CAR, respectively, or T cells without CAR (w/o) were co-incubated with patients’ CLL cells or healthy donor B cells (10⁵ cells each) for 24 hours in an allogeneic setting (CLL cells, n = 11; B cells, n = 4). Alternatively, patients’ T cells were engineered with the respective CAR and incubated with the autologous CLL cells (n = 4). Engineered T cells of healthy donors were coincubated with the respective autologous B cells (n = 4). The CAR T-cell-to-target cell ratio was 5:1. CLL and healthy B cells were labeled with CFSE (0.83 μM) prior to coincubation to discriminate CLL and healthy B cells vs T cells. Staining of CLL and B cells for Annexin V was recorded by flow cytometry. Each dot represents an individual target cell donor. Statistic calculations were performed using the Student t test, *P < .05; ***P < .001; ns, not significant. (C) An example demonstrating killing of CLL cells by modified autologous CAR T cells. T cells from a CLL patient were modified with the respective CARs, and the CAR was recorded 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 (left panel). 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. T cells were cocultivated with autologous CLL cells in a CAR T-cell-to-CLL cell ratio of 5:1 for 24 hours, and the number of apoptotic CLL cells was determined by staining with APC-conjugated Annexin V (right panel). CLL cells were labeled with CFSE prior to coincubation to discriminate CLL cells from T cells. (D) CLL cells harbored an unmutated (n = 4) or mutated (n = 9) immunoglobulin heavy chain variable region (IgVH) status. Patients were in Binet stage A (n = 7), stage B (n = 3), or stage C (n = 5) (Table 2). Anti-FcµR CAR T cells showed an increased killing of CLL cells with an IgVH muted status compared with CLL cells with unmutated IgVH (P = .013). No significant differences in CAR T-cell killing of CLL cells were obtained from patients in different Binet stages. Each dot represents an individual CLL patient. Data represent the mean ± SD.