Fig. 5.

The anti-ROR1 mAb D10 inhibits engraftment of CD5⁺B220^lowROR1⁺ leukemia cells in ROR1 Tg mice. (A) Lysates were prepared from CD5⁺B220^lowROR1⁺ leukemia cells that previously were treated for 1 or 2 h with either 40 μg/mL of control IgG or D10 for immunoblot analysis with antibodies specific for pAKT (Top row), total AKT (Middle row), or β-actin (Bottom row). The numbers between the Top and Middle rows are the ratio of the density of pAKT divided by that of AKT, as in Fig. 4. (B–E) ROR1 Tg mice were engrafted with 5 × 10⁵ CD5⁺B220^lowROR1⁺ leukemia cells and then given weekly i.v. injections of antibody at 10 mg/kg. (B) The blood mononuclear cells of engrafted mice were monitored weekly for CD5⁺B220^low leukemia cells by flow cytometry. Contour plots depict staining of lymphocytes, as determined by light scatter characteristics, with fluorochrome-conjugated mAbs specific for mouse CD5 (y axis) and mouse B220 on the abscissa at 3 wk (Top row), 4 wk (Middle row), or 5 wk (Bottom row) of engrafted mice that were treated with control IgG (Left column), 4A5 (Center column), or D10 (Right column). The percentages in the Top Right of each contour plot indicate the proportion of the blood mononuclear cells having CD5⁺B220^low phenotype of the leukemia cells. (C) Indicated in the graph are the numbers of leukemia cells detected over time in mice treated with D10 (gray), 4A5 (black), or control mIgG (black). (D) Representative spleens of ROR1 Tg mice 4 wk after engraftment with 5 × 10⁴ CD5⁺B220^low B cells and treatment with either control IgG (Left three), 4A5 (Center three), or D10 (Right three) mAbs, as indicated above. For comparison, a representative spleen of a ROR1 Tg mouse that had not been engrafted is to the Far Right (marked normal). (E) Total number of CD5⁺B220^low leukemia cells in spleens of recipient mice 4 wk after adoptive transfer of 5 × 10⁴ CD5⁺B220^low B cells that received weekly injections of 10 mg/kg of mIgG, D10, or 4A5 mAb, as determined by flow cytometric analysis and cell count. Each square represents the number of leukemia cells found in individual mice. The bars represent the mean number for each group ± SEM; n = 3; *P < 0.05, **P < 0.01, compared with the mIgG group, as calculated using the Student t test.