Figure 2.

SPM-2-mediated redirected lysis of blasts from patients with different AML subtypes in combination with NK cells from a healthy unrelated donor. Blasts contained in the PBMC or BMMC populations of AML patients were obtained at the disease stages (diagnosis, remission, relapse) specified in Table 1. Blasts were labeled with calcein and used as targets in RDL reactions mediated by SPM-2 or control proteins in combination with ex vivo expanded, IL-2 stimulated NK cells from an unrelated healthy donor. NK cells were part of a population of LAK cells, consisting to 70% of T cells, 25% of NK cells, and 5% of NKT cells, after ex vivo expansion for 20 d in the presence of IL-2 (Material & Methods). The LAK cells were added in a 10: 1 effector to target cell ratio, corresponding to an effective E: T ratio of NK: targets of 2: 1. SPM-2 triplebody was present in the reactions at the concentrations shown in pM. A) Samples from patients with favorable AML subtype according to the ELN (European Leukemia Network) classification². B) AML with intermediate-I ELN risk subtype. C) Samples from patients with ELN intermediate-II risk subtype. D) samples from patients with adverse ELN risk disease. E) samples from patients with an unclassified disease subtype. F) Myeloid cells from healthy donors (C1, C2), preparatively enriched by immuno-magnetic sorting with CD11b beads show similar susceptibility to SPM-2 mediated lysis as non-enriched blasts from a representative patient sample (C3; patient P1 in Table 1). In all experiments, MOLM-13 cells were carried along as a positive control, and triplebody Her2-16-Her2 as a negative control. Additional controls have previously been performed and reported, showing that target cells devoid of CD33 and/or CD123, such as HEK 293 and CHO cells, failed to bind triplebodies with specificity for CD33 and CD123.⁵⁸ Specific lysis was computed as outlined in Materials & Methods. Error bars represent the standard error of the mean (SEM) computed for triplicate samples of each measurement point.