Fig 2: FcRH5 CAR-T cells specifically recognize and lyse FcRH5+ target cells.a Schematic representation of FcRH5-specific CAR expression construct. b The cytolytic activity of mock T or FcRH5 CAR-T cells against indicated target cells at various effector: target cell (E/T) ratios was determined by a 6-h luciferase-based cytolytic assay. Data represent Mean ± SD from three independent experiments. c Mock T or FcRH5 CAR-T cells were co-cultured with target cells for 24 h, and cell-free supernatants were harvested for evaluating IFN-γ, IL-2 and TNF-α secretion. Mean ± SD, n = 3 independent co-cultures, unpaired two-tailed t-test. d Mock T or FcRH5 CAR-T cells were labeled with eFlour-670 and co-cultured with equal number of target cells for 5 days, and the dilution of eFlour-670 fluorescence signal was determined to reflect cell division. The plots were gated on CD3+ lymphocytes. The above experiments a-d were repeated with 2 different T cell donors. e Flow cytometric analysis of FcRH5 expression on CD138+ primary myeloma cells from three newly-diagnosed MM patients. Solid line represents staining with anti-FcRH5 mAb and dashed line represents staining with isotype control antibody. f The patient-derived myeloma cells were co-cultured with autologous mock T or FcRH5 CAR-T cells for 24 h, and cell-free supernatants were harvested for determination of IFN-γ secretion. Mean ± SD, n = 3 independent co-cultures, unpaired two-tailed Student t-test. g The patient-derived myeloma cells were labeled with eFlour-670 and co-cultured with autologous mock T or FcRH5 CAR-T cells at the E/T ratio of 5:1 for 6 h, and then cytolytic effect of T cells was determined by a flow cytometry-based assay. Mean ± SD, n = 3 independent co-cultures, two-tailed Student t test.

Fig 3: FcRH5 CAR-T cells display tumoricidal activity in the subcutaneous NCI-H929-CBR xenograft model.a Experimental schematic: Male NOG mice aged 6–8 weeks were subcutaneously inoculated with 5 × 106 NCI-H929-CBR cells expressing click beetle red (CBR) luciferase on day −7, and were then intravenously infused with 5 × 106 mock T or FcRH5 CAR-T cells from two different donors (n = 6 mice per group for each donor) when the tumors became palpable (day 0). b Representative tumor bioluminescence of NOG mice at different time points. c Graph showed the quantification of whole-body luminescence in NOG mice from each group at different time points with the lines connecting the mean values. Data shown are representative for results obtained in independent experiments with T-cell from 2 donors. Mean ± SD, n = 6 mice, unpaired two-tailed Student t test, d Kaplan–Meier curve for the overall survival of mice from different treatment group (n = 12 mice per group). Log-rank (Mantel-Cox) test. e The percentage of T cells (gated on human CD45+CD3+) in peripheral blood of NOG mice at different time points was determined by flow cytometric analysis. Data are representative of two independent experiments with T-cell from 2 donors (n = 12 mice per group in total). Mean ± SD, unpaired two-tailed Student t test,.

Fig 4: FcRH5/BCMA bispecific CAR-T cells demonstrate robust anti-MM capacity both in vitro and in vivo.a Cytolytic activity of bispecific CAR-T cells and mono-specific CAR-T cells against indicated target cells was determined by the 6-h luciferase-based cytolytic assay. Mean ± SD, n = 3 independent co-cultures. b Bispecific CAR-T cells or mono-specific CAR-T cells were co-incubated with indicated target cells for 24 h, and cell-free supernatants were harvested for evaluating IFN-γ, IL-2 and TNF-α secretion. Mean ± SD, n = 3 independent co-cultures. Experiments of (a, b) were repeated with two different T cell donors. c Experimental schematic: Male NOG mice aged 6–8 weeks mice were subcutaneously inoculated with 5 × 106 NCI-H929-CBR cells expressing click beetle red (CBR) luciferase on day −7, and were then intravenously infused with 5 × 106 mock T, FcRH5 CAR-T, BCMA CAR-T or FcRH5/BCMA CAR-T cells from two different donors (n = 6 mice per group for each donor) on day 0 when the tumors became palpable. d Representative tumor bioluminescence of NOG mice at different time points. e Graph showed the quantification of whole-body luminescence in NOG mice from each group at different time points with the lines connecting means. Data shown are representative for results obtained in independent experiments with T-cell from 2 donors. Mean ± SD, n = 6 mice per group, One-way ANOVA with Dunnett’s correction for multiple comparison. f Kaplan–Meier curve for the overall survival of mice from different treatment group (n = 12 mice per group). Log-rank (Mantel-Cox) test. g The percentage of T cells (gated on human CD45+CD3+) in peripheral blood of NOG mice at different time points was determined by flow cytometric analysis. Data are representative of two independent experiments with T-cell from 2 donors (n = 12 mice per group in total). Mean ± SD, One-way ANOVA with Dunnett’s correction for multiple comparison.

Fig 5: FcRH5 CAR-T cells induce durable tumor regression in a disseminated MM xenograft model.a Experimental schematic: Male NOG mice aged 6–8 weeks were intravenously inoculated with 5 × 106 MM.1s-FcRH5-CBR cells on day −7, and were then treated with 5 × 106 mock T, FcRH5 CAR-T cells or BCMA CAR-T cells from two different donors (n = 6 mice per group for each donor) on day 0. b Representative tumor bioluminescence of NOG mice at different time points. c Graph showed the quantification of whole-body luminescence in NOG mice from each group at different time points with the lines connecting means. Data shown are representative for results obtained in independent experiments with T-cell from 2 donors. Mean ± SD, n = 6 mice, One-way ANOVA with Dunnett’s correction for multiple comparison. d Kaplan–Meier curve for the overall survival of mice from different treatment group (n = 12 mice per group). Log-rank (Mantel–Cox) test. e The percentage of T cells (gated on human CD45+CD3+) in peripheral blood of NOG mice was determined by flow cytometric analysis. Data are representative of two independent experiments with T-cell from 2 donors (n = 12 mice per group in total). Mean ± SD, One-way ANOVA with Dunnett’s correction for multiple comparison.