Figure 2.
huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ cell lines in vitro. (A) Dose response of ROR1+ pre–B-ALL cell lines Kasumi-2 and 697 to huXBR1-402-G5-PNU (red line) and trastuzumab-G5-PNU (blue line; Tras-G5-PNU). Half maximal inhibitory concentrations (IC50) + 95% confidence interval are as follows: Kasumi-2: huXBR1-402-G5-PNU, 1.270 ng/mL (0.31-5.22); Tras-G5-PNU, 5330 ng/mL (2698-10527); 697: huXBR1-402-G5-PNU, 75.68 ng/mL (50.43-113.5); Tras-G5-PNU, 2987 ng/mL (2463-3622). Viability was measured by using MTS assays, and relative light unit (RLU) values for each dose were generated after 72 hours of treatment in 2 independent experiments. (B) Direct cytotoxicity assay on ROR1+ CLL cell line HG3, MCL cell lines JeKo-1 and Mino, ALL cell line 697, and ROR1– CLL cell line MEC1. 2.5 × 105 plated cells were treated with 10 µg/mL of relevant antibodies and controls. Normalized viability (to vehicle) is reported as measured by Annexin V/propidium iodide staining after 72 hours of culture. n = at least 3 independent experiments. (C) Matched absolute cell count (normalized to vehicle) per microliter of culture for all conditions shown. n = at least 3 independent experiments. (D) Cell cycle analysis as measured by 2-hour AF647 conjugated EdU (AF647) incorporation and FxCycle Violet DNA (BV421) stain in paired samples seen in panel B. G1/G0 (green), AF647–/BV421–; S (blue), AF647+; G2/M (red), AF647-–/BV421+. n = at least 3 independent experiments. For panels C and D: red, huXBR1-402-G5-PNU; green, Tras-G5-PNU; white, unconjugated huXBR1-402; blue, vehicle treatments. (E) Direct cytotoxicity assay on primary CLL purified B cells. 1 × 106 plated cells were treated with 10 µg/mL of huXBR1-402-G5-PNU and relevant antibody isotypes (Tras-G5-PNU; unconjugated huXBR1-402) and controls (vehicle; positive control obinutuzumab). Viability (% live cells) normalized to vehicle treatment is reported as measured by Annexin V/propidium iodide staining after 72 hours of culture. n = 11 patients. All data were analyzed by flow cytometry. Statistical significance for multiple comparisons was analyzed via one-way analysis of variance with Tukey’s post hoc test. Error bars indicate ± SE.

huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ cell lines in vitro. (A) Dose response of ROR1+ pre–B-ALL cell lines Kasumi-2 and 697 to huXBR1-402-G5-PNU (red line) and trastuzumab-G5-PNU (blue line; Tras-G5-PNU). Half maximal inhibitory concentrations (IC50) + 95% confidence interval are as follows: Kasumi-2: huXBR1-402-G5-PNU, 1.270 ng/mL (0.31-5.22); Tras-G5-PNU, 5330 ng/mL (2698-10527); 697: huXBR1-402-G5-PNU, 75.68 ng/mL (50.43-113.5); Tras-G5-PNU, 2987 ng/mL (2463-3622). Viability was measured by using MTS assays, and relative light unit (RLU) values for each dose were generated after 72 hours of treatment in 2 independent experiments. (B) Direct cytotoxicity assay on ROR1+ CLL cell line HG3, MCL cell lines JeKo-1 and Mino, ALL cell line 697, and ROR1– CLL cell line MEC1. 2.5 × 105 plated cells were treated with 10 µg/mL of relevant antibodies and controls. Normalized viability (to vehicle) is reported as measured by Annexin V/propidium iodide staining after 72 hours of culture. n = at least 3 independent experiments. (C) Matched absolute cell count (normalized to vehicle) per microliter of culture for all conditions shown. n = at least 3 independent experiments. (D) Cell cycle analysis as measured by 2-hour AF647 conjugated EdU (AF647) incorporation and FxCycle Violet DNA (BV421) stain in paired samples seen in panel B. G1/G0 (green), AF647–/BV421–; S (blue), AF647+; G2/M (red), AF647-–/BV421+. n = at least 3 independent experiments. For panels C and D: red, huXBR1-402-G5-PNU; green, Tras-G5-PNU; white, unconjugated huXBR1-402; blue, vehicle treatments. (E) Direct cytotoxicity assay on primary CLL purified B cells. 1 × 106 plated cells were treated with 10 µg/mL of huXBR1-402-G5-PNU and relevant antibody isotypes (Tras-G5-PNU; unconjugated huXBR1-402) and controls (vehicle; positive control obinutuzumab). Viability (% live cells) normalized to vehicle treatment is reported as measured by Annexin V/propidium iodide staining after 72 hours of culture. n = 11 patients. All data were analyzed by flow cytometry. Statistical significance for multiple comparisons was analyzed via one-way analysis of variance with Tukey’s post hoc test. Error bars indicate ± SE.

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