BN-CD38 shows potent antileukemic efficacy in vitro and ex vivo. (A) Schematic illustration of the therapeutic rationale to target CD38^neg LSCs with T-cell engagers against CD38 to eradicate AML by uncovering the IFN-γ/CD38 regulatory loop. Graphical representation of (B) BN-CD38 projected structure; (C) distance between T cell and tumor cell membranes upon TCR-MHC interaction; and (D) spatial prediction of how BN-CD38 interaction with T cell (CD3) and tumor cell (CD38) is similar to the interaction observed in the MHC-TCR complex. (E) Surface plasmon resonance (SPR) sensorgrams for CD38 and CD3 binding with different concentrations of BN-CD38. (F) THP1 green fluorescent protein–positive (GFP^pos) cells were cocultured with healthy donor T cells at an E:T ratio of 1:1 overnight (16 hours) in the presence of increasing doses of BN-CD38 active (A) and BN-CD38Mut. THP1 cell killing was determined with 7-aminoactinomycin D staining and gating on GFP^pos cells by flow cytometry. IC₅₀ curves are shown for BN-CD38 and BN-CD38Mut, and data are represented as mean ± standard error of the mean (SEM) of 5 independent healthy donors. (G-H) Samples in panel F were also used to assess the induction of early (CD69) and late (CD25) T-cell activation markers in the total CD4 or CD8 T cells. Dose-dependent T-cell activation curves are represented as mean ± SEM of 3 healthy donors. Ordinary 1-way ANOVA test was used for calculation of statistical significance. (I-K) Total AML cells (n = 7) were treated with 1.0 ng/mL IgG, CD38-NB, BN-CD38 Mut, or BN-CD38 for 5 days and subjected to cell surface staining for CD34 and CD38. (I) Representative flow cytometry contour plots of 2 representative patient-derived AML samples and (J-K) violin plots show elimination of total AML cells (CD34^posCD38^pos blasts and CD34^posCD38^neg LSCs) with BN-CD38 compared with control treatments (IgG, CD38-NB, and BN-CD38Mut). Cell frequencies were normalized to the paired control human IgG treatment and shown as F.C. Overall, 2 of 7 patients were not assessed with BN-CD38Mut and CD38 NB. (L) Total AML cells were treated with 1.0 ng/mL control human IgG, CD38NB, BN-CD38 Mut, or BN-CD38 and plated in CFC assay for 14 days. Violin plot illustrates the normalized CFU F.C. for each treatment group compared with the paired IgG (CD38 NB, n = 4 PB; BN-CD38 Mut, n = 4 PB; BN-CD38, n = 11 [9 PB and 2 BM]). Each dot in each treatment group represents 1 individual patient. (M) Schematic cartoon and representative flow cytometry contour plot of the experimental design and gating strategy used to assess apoptosis of CD38^neg AML blasts by autologous T cells. (N) Contour plots of 2 representative patients with AML showing annexin-V/4′,6-diamidino-2-phenylindole (DAPI) staining of the CD38^neg gated population after treatment with IgG, BN-CD38Mut, or BN-CD38 SNs in the presence of autologous T cells. (O) Violin plot showing changes in induced apoptosis in each treatment group. Unpaired Student t test was used to calculate statistical significance in n = 4 patients with AML; ∗∗∗P < .001, n = 4. For panels G-H,J-L, 1-way ANOVA with multiple comparisons was used to calculate statistical significance between different groups; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. E, effector T cells; ns, not significant; T, target cancer cells.