XmAb5574 and XmAb5603 internalization studies and kinetics of CD19 antibody internalization. (A) CLL cells from 5 patients were incubated with Alexa Fluor 488–labeled XmAb5574, XmAb5603, rituximab, and isotype control IgG1 antibodies, respectively, for indicated times and washed with either acidic glycine buffer to remove externally bound antibody or PBS. FACS was used to analyze the MFI of internalized antibody relative to the isotype (0% control) and no-acidic glycine wash (100% control) conditions by using the formula [MFI experimental − MFI isotype (0% control)]/[MFI 100% control − MFI isotype (0% control)] × 100. The average maximum internalization for XmAb5574 was 27.9% (95% CI, 14.5%-41.4%) at 30 minutes and for XmAb5603 12.2% (95% CI, 0.2%-24.3%) at 120 minutes of incubation. A plateau effect was also observed for antibody internalization at around 30 minutes of incubation (P for trend > .05 for all antibodies tested). (B) Time and dose kinetics of CD19 antibody internalization. B-CLL cells were incubated with Alexa Fluor 488–labeled XmAb5574 (0.2, 1.0, 3.0, or 10 μg/mL) for indicated time periods. The cells were washed with either glycine buffer (100mM glycine, 50mM NaCl, pH 2.7) or PBS buffer (pH 7.4), and the percentage of internalization was determined as described in panel A. (C) Confocal microscopy analysis of uptake of fluorescently labeled CD19 antibodies in B-CLL cells in vitro. B-CLL cells were treated with Alexa Fluor 488 CD19 (XmAb5574 or XmAb5603) for 1 hour or 4 hours. After washing and fixation, the nucleus of cells were stained by DRAQ5. Images are shown in 1× and 4× magnifications. Fluorescence of anti-CD19 (green) and DRAQ5 (blue) were analyzed by using Zeiss 510 META Laser Scanning Confocal Imaging Systems and LSM Image software (Carl Zeiss MicroImaging Inc). (D) XmAb5574 induces minimal direct cytotoxicity in primary patient CLL cells. CLL cells from 9 patients were independently treated with media, goat anti–human IgG (α-Fc) and trastuzumab, XmAb5603, XmAb5574, or rituximab, all with and without α-Fc, and 2-FaraA for 24 and 48 hours, and all at a concentration of 10 μg/mL. Minimal cytotoxicity was observed at 24 or 48 hours (data not shown) after the addition of either XmAb5603 or XmAb5574. Even though XmAb5603 and XmAb5574 antibodies mediated direct cytotoxicity 6.2% (95% CI, 0.1%-11.6%; P = .02) and 7.4% (95% CI, 2.0%-12.7%; P = .02) more than trastuzumab, respectively, this difference may be of limited clinical utility. There was no difference in cytotoxicity mediated by XmAb5574 compared with rituximab in the presence of α-Fc (0.6% increase with XmAb5574; 95% CI, −10.5% to 11.7%; *P = .91). The addition of α-Fc to XmAb5574 also failed to significantly increase its direct cytotoxicity (0.4% increase; 95% CI, −8.9% to 8.8%; P = .99; n = 9; data not shown). The direct cell death at 24-hour points for all cells was assessed by annexin V/PI staining and analyzed by FACS. Percentages of dead cells were calculated as the sum of annexin V⁺ and/or PI⁺ cells, and all values were normalized to media control. Error bars represent SEMs. (E) XmAb5574 does not induce CDC in primary CLL cells. CLL cells at 10⁶/mL were treated with media, rituximab, trastuzumab, XmAb5603, XmAb5574, or alemtuzumab, all at a concentration of 10 μg/mL, in the presence of media, media with 30% plasma from healthy donor blood sample, or media with 30% heat-inactivated (56°C, 30 minutes) plasma for 1 hour. The CDC function was evaluated by PI staining on FACS analysis. Error bars represent SEMs. XmAb5574 does not mediate CDC against primary B-CLL cells (0.8% decrease; 95% CI, −4.5% to 2.9%; P = .99 compared with trastuzumab; and 2.9% decrease; 95% CI, −7.9% to 2.1%; *P = .18 compared with rituximab; n = 3).