In vitro and in vivo antilymphoma activity of anti-HSP105 Ab. (A) DOHH-2 (i), SU-DHL-4 (ii), and NAMALWA (iii) cells were treated with the indicated concentrations of the anti-HSP105 Ab formulation for in vivo use; and after being stained with PI, viable cells in culture (PI⁻) were counted by flow cytometry using a calibrated suspension of fluorescent microspheres at the indicated time points (h indicates hours). The plots show the means of 3 independent experiments. (B) Flow cytometric determinations of apoptosis of the cell cultures described in panel A costained with FITC-labeled annexin V (ANN) and PI. Average percentages of ANN⁻PI⁻ (white), ANN⁺PI⁻ (light gray), ANN⁺PI⁺ (dark gray), and ANN⁻PI⁺ (black) cells in cultures, calculated from the results of 3 independent experiments, are shown. (C) Flow cytometry-based cell-cycle analyses of DOHH-2 (i), SU-DHL-4 (ii), and NAMALWA (iii) cells after 72-hour incubation with anti-HSP105 Ab at the indicated concentrations. Average frequencies of cells in G₁ (black), S (white), and G₂ (gray) cell-cycle phases and SD of 3 independent experiments are shown. (D) SU-DHL-4 (i) and NAMALWA (ii) growth in SCID mice treated with 3 intraperitoneal injections of NaCl solution (ctrl), 250 μg anti-HSP105 Ab, or matched isotype Igs (rabbit IgG) every 4 days (black arrows). Average tumor volumes (measured with calipers) and SE of 2 (SU-DHL-4 model) and 3 (NAMALWA model) independent experiments, in which 6 mice per group were analyzed. Statistically significant differences were calculated by using the 2-way ANOVA: **P < .01; ***P < .001. Black stars represent anti-HSP105 Ab versus rabbit IgG; and gray stars, anti-HSP105 Ab versus ctrl.