Fig. 5: FcγR-mediated effector functions in vitro and B-cell depletion in human whole blood of rituximab IgG1 μtp fusion mAb. | Communications Biology

Fig. 5: FcγR-mediated effector functions in vitro and B-cell depletion in human whole blood of rituximab IgG1 μtp fusion mAb.

From: On-target IgG hexamerisation driven by a C-terminal IgM tail-piece fusion variant confers augmented complement activation

Fig. 5

a CFSE-labelled CLL PBMCs were opsonised with 0.5 μg/ml IgG1 μtp constructs and co-cultured with human MDMs. Phagocytosis was measured by flow cytometry assessing double-positive CFSE and FcγRIII macrophages. Phagocytosis was examined in macrophages skewed in vitro to M0, M1 (Pam3SK4 stimulation), and M2 (IL4/IL13 stimulation) polarisation states. Data show the phagocytic index mean and SD from independent experiments (n = 3). Statistical analysis was calculated using one-way ANOVA. b Calcein labelled Ramos cells were opsonised with rituximab hIgG1 μtp constructs and incubated with freshly purified PBMCs. The calcein release from cells was used to calculate the % of cell cytotoxicity. Individual data points and mean plotted from independent experiments (n = 3). c, d Raji target cells were incubated with either c rituximab hIgG1 μtp fusion mAb or d BHH2 hIgG1 μtp fusion mAb for 24 h at 37°C. DCD was assessed for double-positive annexin-V and PI by flow cytometry. Results show the individual data points and mean from independent experiments (n = 3). Statistics were calculated using two-way ANOVA with repeated measures. e, f Fresh peripheral human blood was incubated with IgG1 μtp fusion mAb (1 µg/ml) for 24 h at 37°C. B-cell depletion (cytotoxicity index [CTI]) was calculated by the ratio of B cells to T cells using flow cytometry. Results show CTI for e rituximab IgG1 μtp fusion mAb and f BHH2 IgG1 μtp fusion mAb. Data are plotted as mean and SD, individual points represent biologically independent donors (n = 12). Statistical analysis was carried out by one-way ANOVA.

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