INCA033989 antagonizes mutCALR-dependent cell proliferation in vivo. (A) Study schematic. NSG mice (n = 4 per group) were inoculated IV with 20 000 Ba/F3-TPOR/CALR^del52 cells. A single 10-mg/kg intraperitoneal dose of INCA033989 or isotype was administered on day 10 after cell inoculation. The number of Ba/F3 cells in the blood, spleen size, and drug exposure were monitored on day 13 (3 days after a single injection of INCA033989). Noninoculated untreated NSG-naive mice were used as controls (see supplemental Methods for details). (B) Relative numbers of Ba/F3 cells in a sample of whole blood. (C) Spleen weight. Data are presented as the mean ± standard error of the mean (SEM; n = 4 mice per group). Significance was calculated using a parametric t test. ∗P < .05; ∗∗P < .01. (D) INCA033989 PK in hFcRn mice. Mice (n = 4) were dosed IV with 5 mg/kg of INCA033989, the drug concentration was measured in the plasma over time, and PK parameters were calculated. (E) INCA033989 does not induce CDC. TF-1-TPOR/CALR^del52 cells were treated with INCA033989 and other control antibodies for 4 hours in the presence of 5% baby rabbit serum for assessment of cell cytotoxicity. Digitonin (10%) was used as the positive control for cell lysis. The graph shows representative results from 3 independent experiments. (F) INCA033989 does not induce ADCC. Jurkat FcγIIIA reporter effector cells and TF-1-TPOR/CALR^del52 cells (4:1 effector-to-target cell ratio) were treated with INCA033989 and other control antibodies for 24 hours for assessment of FcγIIIA engagement. The graph depicts a representative of 3 independent experiments (See supplemental Methods for further details). ADCC, antibody-dependent cellular cytotoxicity; AUC, area under concentration-time curve; Cl, clearance; C_max, maximum observed plasma concentration; ip, intraperitoneal; RLU, relative light units; T_1/2, half-life; Vss, steady-state volume of distribution.