The presentation of WT1 RMF/HLA-A2 complex is measured by binding to tumor cells using mouse ESK2 no. 34 (3 μg/mL). Binding of the ESK2 was ranked as fold increase over isotype control: +, 1.5-fold to threefold increases; ++, >fivefold to 10-fold increases; and −, no binding at all.

HLA-A2 expression was measured by mouse anti-HLA-A2 mAb, clone BB7; +++, >80-fold increases over isotype control; ++, 40- to 80-fold increases; and +, twofold to 10-fold increases.

Cytotoxicity was tested by ESK2 no. 34 BiTE at various concentrations (examples shown in Figure 2). Cytotoxicity mediated by ESK2 no. 34 BiTE was measured by either standard ⁵¹Cr release (5 hours) or luciferase assay (overnight). The killing activity depends on the dosage and the incubation duration as shown in Figure 2. ++, threefold to fivefold increases in the killing over isotype control; +++, >10-fold killing.

WT1 expression of the cell lines used herein were tested for their WT messenger RNA or protein by us and others in previous studies.^9,10,13,16 The data are summarized from 3 to 7 experiments, depending on cell lines.

CML, chronic myeloid leukemia; NHL, non-Hodgkin lymphoma; NT, not tested.