Characterization of the anti-CD22 targeted therapy, moxetumomab pasudotox, for B-cell precursor acute lymphoblastic leukemia

Abstract

Moxetumomab pasudotox is a second-generation recombinant immunotoxin against CD22 on B-cell lineages. Antileukemic activity has been demonstrated in children with chemotherapy-refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL), with variable responses. Here, we report in vitro and in vivo evaluation of moxetumomab pasudotox treatment of human cell lines and patient-derived cells as a preliminary study to understand characteristics of sensitivity to treatment. Binding, internalization, and apoptosis were evaluated using fluorescently tagged moxetumomab pasudotox. Studies in NOD-scid IL2Rg^null mice showed a modest survival benefit in mice engrafted with 697 cells but not in NALM6 or the two patient-derived xenograft models.

Keywords: acute lymphoblastic leukemia; immunotherapy.

FIGURE 1

Differential moxetumomab binding, internalization, and efficacy for BCP-ALL cell lines and patient samples, as labeled at the top. (A) Moxetumomab-AF488 internalization was determined by flow cytometry after indicated incubation time, +/− acidic buffer wash to remove surface-bound antibodies. Red bars, acid-stripped (internalized); blue bars; unstripped (surface + internalized). Cell lines were evaluated as Mean with standard deviation (SD) from two independent experiments with triplicates. The patient-derived leukemia blasts were evaluated with triplicates directly after enrichment process. (b) Confocal images of moxetumomab-AF594 internalization by BCP-ALL cells (red, lower left images in each panel). Cells were incubated with moxetumomab-AF594 for 30 min, followed by acid stripping to remove surface-bound labeled immunotoxin. Leukemia nuclei were stained with DAPI (blue, top left images). Phase contrast images are shown at top right, with merged images in the lower right. Images acquired with 40× objective; scale bars = 5.0 mm. Representative images are shown from three independent experiments. (c) The bar graphs summarize relative frequency of moxetumomab-resistant BCP-ALL cells, determined as Annexin-V negative after 66 hr incubation with moxetumomab-AF594. Data are reported as means with SDs from three independent experiments. (d) NSG mice were injected with BCP-ALL cells (10 × 10⁶, IV) on day 0. Treated groups were administered moxetumomab pasudotox (697 cells, 300 mg/kg; NALM6 and patient-derived cells, 400 mg/kg, IV) for a total of six doses, with the timing indicated by blue arrowheads. Survival was significantly increased in the treated group of 697-xenografted mice (P< 0.0001, Kaplan–Meier log–rank test). Data represent the combined results of two independent experiments with five to six mice for each group (total n = 10–12)